Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
New Delhi-110001
2012
BIOCHEMICAL ENGINEERING: Principles and Concepts, Third Edition
Syed Tanveer Ahmed Inamdar
© 2012 by PHI Learning Private Limited, New Delhi. All rights reserved. No part
of this book may be reproduced in any form, by mimeograph or any other
means, without permission in writing from the publisher.
ISBN-978-81-203-4585-0
The export rights of this book are vested solely with the publisher.
Contents
Foreword xiii
Preface xv
Preface to the First Edition xvii
Biochemical Engineering: A Perspective xix
8. FERMENTATION TECHNOLOGYTRADITIONAL
PROCESSES AND PRODUCTS ............................................... 254272
8.1 Fermentation 254
8.1.1 Anaerobic Fermentation and Products 255
8.1.2 Aerobic Fermentation Processes 263
Summary 271
Exercises 272
Finally, the author has made sure through comprehensive and extensive
coverage of all the relevant topics, inclusive of detailed accounts on
industrial practices, that the interests of young engineers working in this field
are both sustained and taken care of. I am sure this book will be received
well and make noteworthy contributions besides serving its purpose.
I commend and compliment the painstaking efforts of the author.
The main objective of the Third Edition of this book continues to be the
same as that of the previous editions: to cater to the curriculum for a first-
course in biochemical engineering at the undergraduate level of chemical
engineering discipline.
Keeping in mind the course curriculum of BTech Biotechnology and that
offered at BSc Biotechnology, the book has been aptly revamped.
Indeed I have taken this opportunity to upgrade the text by providing
elaborative explanations in certain selected topics. Chapter 7 on Biological
Reactors is extended with the information on membrane bioreactors with
concise description on their configurations and applications. Chapter 9 on
Downstream Processing is given a new dimension by providing detailed
explanations on selected topics, viz. cell disruption, supercritical fluid
extraction, freeze drying, formulation, etc. and with some additions on
microfiltration and nano filtration processes.
A paradigm shift in the question paper pattern has been observed these
days especially by autonomous and deemed institutions by providing a due
weightage of about 2025% to the questions in the form of multiple choice,
fill in the blanks, true/false, etc. in order to test the level of understanding
of the student. Keeping this trend in mind, I have included the so-called
Self-Assessment Exercises in Appendix D, which incorporates a variety of
questions in different forms covering the entire syllabus. Self-Assessment as
an exercise would really be useful for the students who have read the chapters
and understood the concepts thoroughly.
Words shall not suffice to express my gratitude to my father, Dr. S.D
Inamdar who has consistently stood behind me as a mentor in such
endeavours. I also express my sincere gratitude to all my elders who have
always stood by me at times whenever I needed. Many thanks go to my
brothers Sayed Abdul Basith, Dr. Sayed Rehan Ahmar Inamdar and Dr.
Sayed Afaque Hyder Inamdar, who have extended their help in preparing this
manuscript for the third edition.
I am grateful to PHI Learning for publishing this book. Especially,
my sincere thanks go to Babita Misra, Editorial Coordinator and
xv
xvi Preface
Mr. Bijendra Pandey, Copy Editor for helping me with their professional
inputs to further improve the contents of the book.
Finally, I thank the Management of SDME Society, particularly the
Principal and colleagues, SDM College of Engineering and Technology,
Dharwad for their kind support.
xix
xx Biochemical Engineering: A Perspective
trend of developments that have taken place gives us an insight into the
biotechnology engineering industry.
Ø Hierarchy with spotlights: Hybrid antibiotics, recombinant DNA
technology, enzyme immobilization, bioreactors, biofilters, biochips,
and biosensors are some of the spotlights of biochemical engineering.
Hepatitis-B vaccine, thaumatin (protein sweetener and flavour
modifier), alkaloids, steroids, fuel alcohol, etc. are the recent
developments.
Ø The frontiers of biochemical engineering: Conversion of CO2 and N2
into useful synthons, harnessing stem cells for regenerative medicine
and metabolic engineering of complex human diseases.
Ø Fields of application: The present applications of biochemical
engineering are in the areas of production of fine chemicals, alkaloids,
steroids, hormones, fuel alcohol, vaccines, and a host of other
fermentation products.
The principle on which biochemical engineering and biotechnology relies
is: Why trouble to make compounds yourself when a bug will do it for
you?.
In biochemical engineering processes, living organisms are used for
carrying out transformations. Since these living organisms are chemically
complex, they are capable of carrying out transformations which can be
manipulated by controlling their environment or by mutation.
Biochemical reactions are carried out at mild temperatures generally
under 100°C and frequently below 50°C. Nowadays enzymes that are
chemically proteins and water soluble are used as biocatalysts since they are
powerful and action specific, although the cost for their isolation is
exorbitant. Also, cells, microbes and enzymes are used in biochemical
conversions.
Relationship between Biological Sciences, Processes and Chemical
Engineering
We see from the following table that there exists a relationship between
biological sciences, processes and chemical engineering that amalgamate to
form a biochemical process industry. The interdisciplinary interaction taking
place today between microbiologists, biochemists, and chemical engineers is
also explained.
Organism Selection
Sterilization
Process Control Formulation
Processing
xxii Biochemical Engineering: A Perspective
Microbiology Fundamentals
1
2 Biochemical Engineering: Principles and Concepts
On the other hand, there are certain microbes which are harmful to
human beings. For instance, these microbes can cause diseases, spoil food,
and deteriorate materials like iron pipes, lenses, and wood pilings. On the
large scale of the biosphere, which consists of all the regions of the earth
containing life, microorganisms play an important role in capturing energy
from the sun. Their biological activities also complete the critical segments
of the cycles of carbon, oxygen, nitrogen, sulphur and other elements
essential for life.
Most of the microorganisms are unicellular, i.e. they have a single cell
and all the life processes are performed by it. In the so-called higher forms
of life, i.e. multicellular organisms, cells are in plenty and are arranged in
tissues and organs performing specific functions. Irrespective of the cell
complexity of an organism, the cell is the basic structural unit of life. All the
living cells are fundamentally similar.
It was Robert Hooke (16351703), an English, who first coined the word
cell. The concept of the cell as the structural unit of life was given by two
Germans, Schleiden and Schwann in the year 183839.
The cell: It says:
I am the unit of biological activity;
Organized into the subcellular organelles;
Assigned to each other are the specific functions and duties;
Thus, I truly represent life!
All the living systems have the following characteristics in common:
1. The ability to reproduce.
2. The ability to ingest or assimilate food substances and metabolize
them for energy and the growth.
3. The ability to excrete waste products.
4. The ability to react to the changes in their environment, sometimes
called irritability.
5. The susceptibility to the sudden changes in the genetic constitution,
called the mutation.
In microbiology, we can study microorganisms in great detail and
observe their life processes while they are actively metabolizing, growing,
reproducing, aging and dying.
By modifying their environment, we can alter metabolic activities,
regulate growth, and even change some details of their genetic patternall
without destroying the organisms.
Microorganisms have a wider range of physiological and biochemical
potentialities than all other organisms combined. For example, some bacteria
are able to utilize atmospheric nitrogen for the synthesis of proteins and
other complex organic nitrogenous compounds. Other species require
inorganic and organic nitrogen compounds as initial building blocks for their
nitrogenous constituents. Some microorganisms synthesize all their vitamins,
while others need to be furnished vitamins.
Microbiology Fundamentals 3
KINGDOM FUNGI
Absorption
Photosynthesis Ingestion
KINGDOM PROTISTA
KINGDOM MONERA
2. They can accept a wide variety of nutrients and select the best among
them.
3. They are versatile.
Description of a prokaryotic cell: Figure 1.2 shows a micrograph of a
prokaryote.
%GNN/GODTCPG
Cell membrane 4KDQUQOGU
%GNN9CNN Ribosomes
Cell wall
Nuclear zone
0WENGCTTGIKQP
Figure 1.2 Electron micrograph of a prokaryote.
8CEWQNG
)QNIK 'PFQRNCUOKE
EQORNGZ
TGVKEWNWO
.[UQUQOG
0WENGWU
4KDQUQOGU
/KVQEJQPFTKC
Flow Chart
6KUUWGJQOQIGPCVG
5VTCKP
%GPVTKHWIGHQTOKPCVI
0WENGKCPF%GNNU
%GPVTKHWIGUWRGTPCVCPVHQT
OKPCVI
/KVQEJQPFTKCCPF.[UQUQOGU
%GPVTKHWIGUWRGTPCVCPVHQT
Centrifuge supernatant for
JTCVI
1 hr at 100,000 g
'PFQRNCUOKETGVKEWNWOCPF4KDQUQOGU
5WRGTPCVCPVJCUCUQNWDNGRQTVKQPQHE[VQRNCUO
Figure 1.4 shows the process of differential centrifugation.
Homogenate
Centrifuge Supernatant
supernatant contains soluble
for 5 min at portion of
15,000 g cytoplasm
Strain
Centrifuge
supernatant
for 1 hr at
100,000 g
a b c
Centrifuge a b c
for 10 min
at 600 g
Figure 1.4 Differential centrifugation: (a) nuclei and broken cells, (b) mitochondria and
lysosomes, and (c) endoplasmic reticulum and ribosomes.
1.6.1 Protists
The kingdom protista consists of all the unicellular organisms and also the
10 Biochemical Engineering: Principles and Concepts
organisms containing multiple cells which are of the same type. In short, it
consists of all living things with simple biological organization.
Classification: The kingdom Protista has been classified taking into
consideration the difference in the energy and nutritional needs, growth,
product release rates, reproduction methods, capability of movements and of
course the morphological factors. Refer to Figure 1.5.
Kingdom Protista
Prokaryotes Eukaryotes
Bacteria Cyanobacteria
Moulds Yeasts
1.6.2 Bacteria
Bacteria are very small organisms which are enclosed in cell walls. The outer
surface of the cell wall is covered by a slimy, gummy coating called the
capsule or slime layer. These organisms are unicellular and motile. They
reproduce by division process called the binary fission, through which the
organisms divide into two daughter cells.
Gram reaction: It is the response of the bacteria to a rapid staining test.
Here the cells are stained with a dye crystal violet, then treated with iodine
solution and finally washed in alcohol.
Bacteria which retain blue crystal violet colour after the test are Gram
positive and which lose colour after the test are Gram negative. Figure 1.6
depicts a summary of morphology of Gram-positive and Gram-negative
bacteria.
Commercial usage: Commercial usage of bacteria is based on the oxygen
supply, i.e. whether it is by aerobic or anaerobic process.
Microbiology Fundamentals 11
Capsule or slime
Cell membrane
Protein
components
Cell wall
Mucocomplex
substances
h a b c d e a. Capsule
f b. Cell wall
c. Cytoplasmic
membrane
d. Nucleus
e. Lipid
g f. Flagellum
g. Sulphur vacuole
i h. Cell sap
1.4 micro mt i. Glycogen
Bacteria are the simplest form of plant life. They are single celled, use
soluble food and are capable of reproduction. They are the fundamental
organisms that stabilize organic wastes. Bacteria are seen in huge numbers
in sewage (1 litre of sewage may contain 2225 millions of bacteria).
Majority of bacteria are harmless to human life and are used in many
beneficial processes, for example, in conversion of complex organic compounds
of sewage to stable, organic and mineral compounds resulting in purification.
On the contrary some of the bacteria are pathogenic to human life.
Bacteria are also classified on the basis of their nutritive needs such as
heterotrophic and autotrophic bacteria.
Heterotrophic: This class of bacteria use organic compounds and
carbon for synthesis. There are four categories in it. They are aerobic which
depends on free oxygen; anaerobic, which do not depend on free oxygen;
facultative, that are flexible; and saprophytic, which depends on the dead
organic matter and are found in the upper layers of soil.
Autotrophic: This class of bacteria use CO2 as carbon source and
oxidize inorganic compounds for energy. Nitrifying, sulphur and iron bacteria
are autotrophic bacteria. These bacteria are responsible for corrosion in sewer
systems.
Three forms of bacteria: As we know that bacteria are unicellular, but they
exist in three basic morphological forms such as:
· Spirilla or Spirals
· Cocci or Spheres
· Bacilli or Rod like
Figure 1.8 shows these three forms of bacteria.
Note: Bergeys classification and other selective forms of bacteria are treated in
Appendix C.
Microbiology Fundamentals 13
Spirilla or Spiral
Spirilla or spiral shaped
Cocci orSphere
Cocci or sphere shaped
shaped
Bacilli
Bacillioror rod shaped
Rod shaped
1.6.3 Yeasts
Yeasts form an important group of fungi. They are widespread in nature,
reside in soil of low humidity and cannot trap energy from sunlight.
They measure 530 µm long and 15 µm wide. The modes of reproduc-
tion seen in yeasts are budding (most common), fission and sporulation.
Budding
Fission
a
Ascospores
Diploid
a a a a
Sporulation a
The yeast cycle: Figure 1.12 depicts the life cycle of yeast.
Offspring
Parent cell
Budding
Fission a
Diploid
a a
Conjugation
a
Sporulation
a
a
a Germination aa
a Zygote
1.6.4 Moulds
Moulds are a higher class of fungi with a vegetative structure known as
mycelium (Figure 1.13).
Cytoplasm
Hyphae
Hyphae
Asexual spores
Hyphae
Penicillium
Aspergillus niger
Light
CO2 + H2O CH2O + O2 + H2
(New algae cells)
Algae are found usually in the oxidation ponds. The most common algae
are Chlorella (green algae).
In Japan, algae food cultivation is done and are also used as
foodstuffs and food supplements.
Protozoa: They are strictly aerobic, non-photosynthetic organisms which
reproduce by fission process. They are found in oxidation ponds and are used
are used in water treatment processes as trickling filters, activated sludge
processes and also as biological control agents. Rhizopoda, Flagellate, Ciliate,
etc. are common protozoa.
Viruses: Since viruses are acellular and possess both living and non-living
characteristics, they are considered neither prokaryotic nor eukaryotic. They
Microbiology Fundamentals 17
Atmospheric oxygen
and in water
Carbon dioxide
Figure 1.15 Oxygen cycle.
Industries Plants
Bacteria
Animals
SO4
Death and decay
H2S
H2S
Phosphate rocks
Guano deposits Bacterial action
Marine sediments
Phosphate in H2O Living organisms
Pool of nitrogen in
atmosphere
Animals
Free nitrogen-fixing
bacteria
Man
Decomposition
Egg
Sedimentation Plankton
Herbivores Carnivores
SUMMARY
In this chapter, we learnt the following:
· The cell is a structural and functional unit of life. An eukaryotic cell
consists of well-defined subcellular organelles, enveloped in a plasma
membrane.
· The nucleus contains DNA, the repository of genetic information.
· Mitochondria are the centres for energy metabolism. They are the
principal producers of ATP, which is required for cellular work.
· Endoplasmic reticulum is a network of membranous tubules that
extend throughout the cytoplasm. Ribosomes are the sites of protein
synthesis. Golgi complex is the cluster of membrane vesicles to which
the newly synthesized proteins are handed over for further processing
and export. Lysozomes are the digestive bodies of the cell, actively
involved in the degradation of cellular compounds.
· Cell fractionation or cell differentiation is a method used for the
isolation and separation of cell organelles based on the density
differences using an ultracentrifuge.
· Gram staining test used for the identification of bacteria as Gram
positive and Gram negative.
· Microbes can grow in extreme conditions: at temperatures above
boiling and below freezing. Prokaryotes are primitive organisms and
are usually single celled.
· Bacteria, yeasts, moulds, algae, and protozoa are the industrially
exploited microorganisms.
· Endospores are the dormant forms of bacteria that can resist adverse
conditions.
· Yeasts reproduce asexually by budding, fission, and sporulation.
· Nitrogen, carbon, sulphur, phosphorus, and calcium cycles are called
biogeochemical cycles.
EXERCISES
1.1 What are the characteristics or features that all the living things have
in common?
1.2 Compare prokaryotes and eukaryotes in terms of internal structure
and functions.
1.3 Write on the Whittakers five-kingdom concept.
1.4 Give a broader classification of microorganisms belonging to the
kingdom of Protists.
1.5 Describe the internal structure of a bacterial cell.
Microbiology Fundamentals 27
1.6 With a neat diagram, explain all the sequences of steps involved in the
process of cell fractionation or cell differentiation.
1.7 Describe the important cell types.
1.8 Explain briefly the following terms with their significance:
(a) Cell wall
(b) Nucleus
(c) Chloroplast
(d) Plasma membrane
(e) Mitochondria
1.9 Describe the yeast cycle of reproduction.
1.10 What are the uses of yeasts?
1.11 Describe the mycelial structure of moulds and the applications of two
important industrial moulds.
1.12 Brief the distinct features of actinomycetes and their important
products.
1.13 Compare protozoa with algae in terms of their cellular structures and
functions.
1.14 What are the three forms of bacteria? Explain the process of Gram
staining test. How is it used in classifying the bacteria?
1.15 Explain the process of formation of endospores in bacteria.
1.16 Write notes on the following:
(a) Industrial microbiology
(b) Environmental microbiology
1.17 Describe the following biogeochemical cycles:
(a) Sulphur cycle
(b) Nitrogen cycle
(c) Phosphorus cycle
(d) Carbon cycle
(e) Calcium cycle
(f) Oxygen cycle
Chapter 2
Biological Polymers
Much of the chemistry of the cell is common to all living systems and is
directed towards ensuring growth and cell multiplication, or at least the
survival of the cell. Organisms also share various structural characteristics.
They all contain genetic material, membranes, cytoplasm, etc. All these are
supported by polymeric chemicals called biopolymers.
This chapter deals with predominant cell polymeric chemicals and
smaller monomer units from which larger polymers are derived. It covers
biopolymers, viz. proteins, carbohydrates, lipids and fats, nucleic acids,
enzymes and to some extent, trace elements, micronutrients and macro-
nutrients of the cell, vitamins, steroids, etc. The importance of these
biochemicals is also discussed. At the end of the chapter is given the
hierarchy of biological structure that signifies the proliferation right from the
environmental precursors to the organism.
2.2 LIPIDS
Lipids are regarded as the biological substances or compounds that are
relatively insoluble in water and soluble in organic solvents such as alcohol,
ether, benzene, chloroform, etc. They are related to the fatty acids and used
by the living cells. Fatty acids (saturated) are simple lipids with the following
general formula:
CH3(CH2)n C = O
|
OH
where n = 1220.
The hydrocarbon chain has two identical carbon monomers and
therefore are called the non-informational biopolymers. At the end there is a
carboxylic group, for example, stearic acid for which the value of n is 16.
Unsaturated fatty acids are formed by replacing of a saturated bond by a
double bond, for example, oleic acid.
Complex lipids (Compound lipids): These lipids are the esters of fatty acids
with alcohols having additional groups as phosphates, nitrogenous base,
carbohydrates, proteins, etc. For example:
1. Phospholipids, which are the fatty acids with alcohols and a
phosphorus group. Examples: lecithin, cephalin, etc.
2. Glycolipids, which have a carbohydrate with a nitrogenous base.
Examples: cerebrosides, gangliosides, etc.
3. Lipoproteins are the macromolecular complex of lipids with proteins.
Example: amino lipids.
Derived lipids: These lipids are obtained by the hydrolysis of simple and
complex lipids, for example, lipid soluble vitamins, steroids, etc.
Miscellaneous lipids: Carotenoids, squalene, hydrocarbon as pentacosane
(in beeswax), terpenes, etc. all belong to the class of miscellaneous lipids.
Neutral lipids: These are the uncharged particles and behave neutral.
Cholesterol is the best example.
Non-polar Tail
Polar
Head
Tails
Air
Heads Interface
Water
Figure 2.2 Lipid monolayer formation.
Aqueous phase
Non-polar phase
Aqueous phase
Figure 2.3 Lipid bilayers.
resultant, i.e. micelle and the solution, is lower than that of the original
solution.
This process is important in the digestion of lipids and absorption as
well. The micelle formations increase the number of hydrophobic
hydrophobic and hydrophilichydrophilic interactions or contacts and
subsequently decrease in the hydrophobichydrophilic associations. Figure
2.4 shows a lipid micelle.
Non-polar
phase
Head
Aqueous phase
Tail
C D
A B
2.3 PROTEINS
Proteins (from the Greek word proteios, which means primary) are the
most abundant organic molecules of the living system. They occur in
every part of the cell and constitute about 50% of the cellular dry weight.
Proteins are the high molecular weight substances, nitrogen rich, and
present in all animals and plants. They are composed of 20 amino acids
and are organized into primary, secondary, tertiary and quaternary
structures.
Dynamic functions: These are the most diversified in nature and include the
proteins acting as enzymes, hormones, blood clotting factors, immunoglo-
bulins, membrane receptors, storage proteins, in genetic control, muscle
contraction, respiration, etc.
These proteins are regarded as the working horses of the cell. The
proteins and their expressions represent the functional form of DNA informa-
tion.
PROTEINS
R R
H C NH2 NH2 C H
COOH COOH
D-amino acid L-amino acid
CH3
Phenylalanine — (CH2)3NHC.NH2 Arginine (Arg)
CH2 (Phe) +NH2
O
— CH2C Asparagine (Aspn)
NH
O
— CH2CH2C Glutamine (Glun)
NH2
Biological Polymers 39
2JG )N[
8CN .KG
#UP
8CN
)NP
)NW
*KU
)NP
.GW
5GT
%[U #NC
%[U S S
)N[
5GT
*KU
.GW #NC
8CN )NW
Figure 2.11 The amino acid sequence in the insulin molecule of oxen.
lowering the free energy of the molecules and their interactions with water.
Generally, these interactions occur over relatively short range between different
parts of the molecules.
Figure 2.12 illustrates the protein secondary structure with different
configurations.
C
C C N
N
C C
C C N
C
C N C
C
N
N C
C
N
N C
C
C
N
C N C
C
C C
N
N C
C C N C
(i) Hydrogen bonding (ii) Ball and stick (iii) Ribbon structure
structure structure
(a) a-helix structures
C C C Up
C N C
N C N
C C C Down
C N C
N C N
C C C Up
C N C
N C N
(i) Hydrogen bonding (ii) Ball and stick (iii) Ribbon structure
structure structure
(b) b-pleated sheet
Figure 2.12 Secondary protein structure.
A B C
Ionic attraction
D E F
4CPFQOEQKN
Random coil
0CVKXGHQTO
Native form
&GPCVWTCVKQP
Denaturation
4GPCVWTCVKQP
Renaturation
Native form
0CVKXGHQTO
Figure 2.14 Protein denaturation and renaturation.
Base
OH
HO P O CH2 O
O
H H
H H
OH OH
Ribonucleotides
(general structure)
H H DNA and RNA
N O
H
N N
N N
H H H
N N H N N N
H Purines H H
Adenine Guanine
DNA only H
RNA only
H O
O N
H3 C H H H H
N N N
H N O H N O H N O
H H
H
Thymine Cytosine Uracil
Pyrimidines (Nitrogenous bases)
Figure 2.15 Structure of nucleotides and nitrogenous bases.
Biological Polymers 45
DNA RNA
Figure 2.16 Covalent backbone of nucleic acids.
46 Biochemical Engineering: Principles and Concepts
P H OH
N H
N H O CH3
N
N H N H
N
N
Deoxyribose O
H Deoxyribose
A T
P H P
H N O H N H
N N H N
N N
Deoxyribose
N H O Deoxyribose
G C
2 nm
P
D
P P
D C G
P
D A T D
P P
D T A D
P
G C D
P P
D
P P
A T D
P
D G C D
P P
D T A D
P
D C G
P P
D
P P
3.4 nm D A T
P
D A T D
P P
D G C D
P
A T D
P P
D
P P
C G D
P
D T A D
P
Figure 2.18 Double-helix structure of DNA.
Both the strands of the helix form right-handed coils, but they are
antiparallel to each other, the 5¢ end of one strands facing the 3¢ end of the
other and vice versa.
Chargaffs findings suggest that the number of adenine and guanine
residues equals respectively those of thymine and cytosine residues in a DNA
double-helix. It also follows that purines equal pyrimidines in number in the
molecule, and that the two strands are complimentary to one another in base
sequence.
48 Biochemical Engineering: Principles and Concepts
5¢ 3¢ 5¢ 3¢
Parent
Replica
5¢ 3¢
Guanine
Cytosine
Adenine
5¢ Thymine
3¢
Figure 2.19 DNA replication.
Parental DNA
Ist Generation
2nd Generation
Based on the findings of Watson and Crick, the central dogma of the
molecular genetics is understood and the flow of information is seen to be
essentially in one direction from DNA to the protein.
Three major steps are defined in the flow of information. They are
replication, transcription and translation of the genetic material. The same
is shown in Figure 2.21.
Biological Polymers 51
Transcription
Translation
2.8 CARBOHYDRATES
Carbohydrates are the most abundant organic substances in nature. Sugars,
starches and cellulose found in green plants, glycogen in animal tissues, and
glucose in the body fluids of animals are all examples of carbohydrates.
They occur in food, wood, paper and synthetic fibres. Carbohydrates
have an empirical formula Cn(H2O)n where n ³ 3. They are also called
saccharides meaning sugars.
Carbohydrates play a key role as storage and structural compounds in
the cell. They also play crucial roles in modulating aspects of chemical
signalling in animals and plants. Carbohydrates are synthesized by plants
through photosynthesis as illustrated in Figure 2.22.
CO 2 + H 2O
Photosynthesis
CH2O + O 2
Respiration
Energy released
Figure 2.22 Synthesis of carbohydrates.
CO2 and H2O are converted through photosynthesis into sugars in the
presence of sunlight and are then polymerized to yield polysaccharides.
Carbohydrates are classified into (a) monosaccharides, (b) disaccharides,
and (c) polysaccharides.
2.8.1 Monosaccharides
Monosaccharides are the simplest and smallest carbohydrates and contain
three to nine carbon atoms, and are the building blocks of complex
carbohydrates. These cannot be hydrolyzed into a simpler sugar, for example,
glucose which has a molecular formula of C6H12O6.
Biological Polymers 53
H OH
CHO C
HCOH HCOH
OHCH OHCH O
HCOH HCOH
HCOH HC
CH2OH CH2OH
D-Glucose,
D-Glucose,linear
linear D-Glucose
D-Glucoseringring
structure
structure structure (pyranose)
structure (pyranose)
Figure 2.23 Structure of glucose.
CH2OH CH2OH
O O OH
OH OH
OH OH OH
OH OH
a-D-Glucose b-D-Glucose
Figure 2.24 Structures of a-D-glucose and b-D-glucose.
Tetroses (C4H8O4): Tetroses like erythrose are rare in nature and occur
mainly in bacteria.
5 CH
CH22OH 5 CH22OH
OH
O O
4 1 4 1
3 OH 3 OH
2 2
OH OH OH
D-Ribose Deoxyribose
Figure 2.25 Structures of ribose and deoxyribose.
2.8.2 Disaccharides
These are formed by the condensation of two monosaccharides usually
hexoses. On hydrolysis, a disaccharide yields the two respective
monosaccharides.
glucose + fructose ® sucrose (cane sugar)
glucose + galactose ® lactose (milk sugar)
glucose + glucose ® maltose (in germinating seeds)
Maltose is formed by the condensation of two glucose molecules via
a-1,4glycosidic linkage, which is shown in Figure 2.26.
Biological Polymers 55
CH2OH
CH 2 OH CH2OH CH OH CH OH
CH 2 OH CH22OH CH22OH
O O O O
1
4 +H
H22OO
OH OH OH O OH
OH OH OH OH OH OH
OH OH OH OH
a-D-Glucose a-Maltose
Figure 2.26 Formation of a-maltose.
In the same way, sucrose and lactose synthesis are also shown in
Figure 2.27. Sucrose is a disaccharide of a-D-glucose, and b -D-fructose, and
lactose is a disaccharide of b -D-glucose and b -D-galactose.
CH2OH
CH2OH
O O
OH OH
OH O
OH CH2OH
OH OH
a-D-Glucose b-D-Fructose
(a) Sucrose
CH2OH OH
O
O OH
OH
OH O OH
OH CH2OH
b-D-Glucose b-D-Galactose
(b) Lactose
Figure 2.27 Synthesis of sucrose and lactose.
2.8.3 Polysaccharides
Polysaccharides are normally amorphous, insoluble in water and are
tasteless. These are referred to as non-sugars. Polysaccharides are composed
of ten to many thousands of monosaccharides in their macromolecules, and
their empirical formula is (C6H10O5)n.
Polysaccharides are formed by the condensation of more than two
monosaccharides by glycosidic linkages or bonds. Thus, these are the
56 Biochemical Engineering: Principles and Concepts
C C
O O
O
O O
O
C CH2 C
O O O
O O O
O O O O
C C C
O O O
O
O O O O
Dextrans: These are produced by yeasts and bacteria and are made up
of glucose residues linked mainly by 1 ® 6 a glucosidic bonds. Branches are
formed by occasional 1 ® 4 a linkages and rarely through 1 ® 3 aÿlinkages.
They have the property of absorbing water to form viscose colloidal solution.
They are not metabolized by the tissues and hence are used for retaining
water in circulation for a long period by administering it intravenously
(plasma volume extender).
Inulin: Inulin is a low molecular weight polysaccharide present in
tubers. It is a polymer of fructose. Not being utilizable, it is used in assessing
the glomerular filtration rate (GFR) in the study of human kidney function.
Agar: Agar is found in sea weeds. It consists of two main components:
agarose and agaropectin. Agarose is a non-sulphated linear polymer consisting
of alternating residues of D-galactose and 3,6-anhydro-L-galactose.
Agaropectin is a mixture of sulphated galactans, which may also contain
glucoronic acid or pyruvic acid. It dissolves in hot water and sets to gel on
cooling. It is extensively used for culturing the microbes.
Chitin: Chitin forms the characteristic exoskeleton of invertebrates
like crab, lobster, prawns, etc. It is a polymer of acetylglucosamine.
Pectin: Pectins are present in apples, lemon and other fruits. In the
middle lamella of cell wall, pectin is found as calcium pectate. Pectins
consist of repeating units of galactose and galacturonic acid.
Heteropolysaccharides: Let us learn more about the following
heteropolysaccharides:
Hemicellulose: It is found in association with cellulose in cell walls.
The commonly found sugars in hemicellulose are D-xylose, L-arabinose,
D-galactose, and D-glucoronic acid.
Gums: Gums are substances exuded by plants on mechanical injury or
after bacterial, fungal or insect attack. The viscose substance helps to seal the
wound and protects the plants. The polysaccharides in these are highly branched.
Hyaluronic acid: Hyaluronic acid is the simplest mucopolysaccharide
and linear polymer of disaccharides which form repeating units. Each
disaccharide is linked to the next byÿb-1, 4-glucosidic bonds. It consists of two
monosaccharides D-glucoronic acid and N-acetyl-D-glucosamine. It is found
in the skin, vitreous humour of the eye, the umbilical cord, as a coating
around ovum and in certain bacteria.
Heparin: Heparin is an anti-coagulant secreted by most cells in the
intestinal mucosa, liver, lung, spleen and kidney. It is a polymer of
glucoronic acid and N-acetylglucosamine.
58 Biochemical Engineering: Principles and Concepts
Organism
Organs
Tissues
Cells
Multienzyme complexes,
ribosomes, chromosomes,
Supramolecular assemblies
contractile systems
(MW: 1061010)
SUMMARY
In this chapter, we learnt the following:
· Lipids are the compounds of critical importance in the construction
of cellular membranes.
· Fats serve as the reserves of energy and a number of growth factors
or hormones involve lipid materials. Glycolipids and phospholipids
are the primary components of the biological membranes.
· Lipids exhibit stable configurations like lipid monolayer, lipid bilayer
and lipid micelle.
· Proteins perform structural and dynamic functions in the cells and
organisms.
· Proteins are the polymers made up of L-a-amino acids which
are 20 in number and are classified into different groups based on
their structure, chemical nature, nutritional requirements and
metabolism.
· Amino acids have two functional groups, namely carboxyl (COOH)
and the amino (NH2) and they exist as dipolar ions commonly called
zwitterions.
· The structure of proteins is divided into four levels of organization,
namely primary, secondary, tertiary and quaternary. The primary
structure represents the linear sequence of amino acids, the secondary
is the twisting and special arrangement of polypeptide chain, the
tertiary is the three-dimensional structure of a functional protein, and
the quaternary structure represents the assembly of similar or
dissimilar subunits.
· The tertiary and the quaternary protein structures are stabilized by
non-covalent hydrogen bonds, hydrophobic interactions and ionic
bonds.
· DNA is the chemical basis of heredity organized into genes, the basic
units of information.
· All the three RNAs are produced by DNA and carry out the synthesis
of proteins.
· Nucleic acids are the polymers of nucleotides called polynucleotides,
which are held by phosphodiester bridges. A nucleotide is a
combination of base + sugar + phosphate.
· Nucleotides perform a wide variety of cellular functions as energy
carriers, metabolic regulators, etc.
· The structure of DNA is a double helix (Watson and Crick model)
composed of two anti-parallel strands of polydeoxyribonucleotides
twisted around each other. The strands are held by the hydrogen
bonds between the nitrogenous bases.
Biological Polymers 61
EXERCISES
2.1 What are repetitive and non-repetitive biological polymers? Give
some examples for the same.
2.2 Explain saturated and unsaturated fatty acids. Discuss with neat
sketches the stable configurations of fatty acids in water.
2.3 Enlist the functions of lipids and fats.
2.4 What is the general steroid base? Give its structure.
2.5 Write a note on fat soluble vitamins and steroids.
2.6 Describe the primary, secondary, tertiary and quaternary structures of
proteins.
2.7 Describe the Watson and Crick model of DNA.
2.8 Write on the following:
(a) DNA replication
(b) Biological Information Storage
(c) Types of RNA and their functions
(d) Comparison between DNA and RNA.
2.9 Define carbohydrates. Explain the formation of these carbohydrates in
nature.
2.10 What are simple sugars? With examples, explain the synthesis of the
various types of monosaccharides.
2.11 What are the monosaccharides that are commonly found in the
biological systems?
62 Biochemical Engineering: Principles and Concepts
63
64 Biochemical Engineering: Principles and Concepts
Mountain
Tunnel
Overall it is seen that catalytic reactions are much faster than the non-
catalytic reactions. The standard free energy change remains unchanged in
both the cases.
These mechanisms are well explained by the Arrhenius equation, i.e.
K = AeEa/RT (3.1)
where A = Arrhenius constant
Ea = energy of activation
R = gas constant
T = temperature
';0=
V K ;3= (3.2)
'T
where k is the rate constant that indicates the speed or the efficiency of a
reaction. If we look at the graph of v vs [S], it is linear (Figure 3.2).
Velocity of reaction, v
The overall kinetic order of a reaction is the sum of the exponents in the
equation, i.e. as to how many molecules are reacting in the rate-determing
step. Therefore, S ® P is a first order reaction.
Now, consider
S1 + S2 ® P1 + P2
It is a single step reaction. In this case, the rate is found by knowing the
concentrations of both the reactants, i.e. the substrates. If S1 and S2 are
present in similar concentrations, the rate equation is
v = k[S1]1[S2]1
With respect to each reactant, the reaction is the first order and overall
it is a second order or a bimolecular reaction.
If [S2] ? [S1], so that it remains essentially constant, the reaction is a
zero order reaction with respect to that reactant, i.e. [S2]. So [S2] is
unchanging and can be eliminated. Therefore,
v = k[S1]1[S2]0
or v = k¢[S1]
For example, in the non-enzymatic hydrolysis of sucrose in the aqueous
acid solutions,
C12H22O11 + H2O C6H12O6 + C6H12O6
68 Biochemical Engineering: Principles and Concepts
Here [H2O] ? [S1] and [H2O] is constant. So the rate depends only on the
concentration of sucrose, i.e. [S1].
Now let us consider purely enzymatic reactions and the various mecha-
nisms of interaction of substrates and formation of products.
Generally, an enzymatic reaction is given as follows:
E + S
ES
E + P
where
E = enzyme
S = substrate
ES = enzymesubstrate complex
P = product
The prime requisite for the enzyme catalysis is that S must combine with
E at the active site to form ES, the enzymesubstrate complex. Once
combined, after sometime form the products with the liberation of E.
More specifically, it is represented as
E + S ES
E+P
which shows that the formation of ES complex is reversible and the formation
of the products is irreversible. The idea of ES complex formation was first
conceived by MichaelisMenten in the year 1913 and has been widely accepted.
A few theories have been proposed to explain the formation of ES complex.
Emil Fischer proposed that E is a rigid template and S is a matching key.
The rate of enzymatic reactions depends on the concentration of both the
substrate and enzyme. When the amount of S is high enough or when the
amount of E is much less than the S, then the reaction is the pseudo first
order, i.e.
v = k[E]1[S]0
or v = k[E]1
or v = k¢[E]
It shows that the more is enzyme present, the faster is the reaction.
Figure 3.3 shows that with increase in the concentration of enzymes the rate
increases.
Velocity, v
Note: [S] is high and fixed, i.e. at saturation throughout the reaction.
Pseudo first order reactions are used in the analysis called enzyme assays
that determine the concentration of enzymes.
2E
[P]
concentrations. The lines drawn to the curve indicate the initial period, which
is at the lower portion of the curve and given as
v0 = D[P]/Dt (slope of the curve) (3.3)
As the reaction proceeds, [P] increases. The rate of reaction doubles when
twice as much enzyme (2E) is added to another identical reaction mixture.
K
Z K CAT
ZZZZX
% 3 YZZZ Z %3
Z %0
K
ES
E
(a) Lock and key model
ES
E
(b) Induced fit model
ES
E
(c) Substrate strain theory
Figure 3.5 Common configuration for ES complex formation for depicting the three theories.
K K
ZZZZX
% 3 YZZZZZ
Z %3
% 0
K
where k2 = kcat. According to the slow rate determining step, the rate of
reaction is given as
D;3= D;0=
R K ;%3= (3.4)
DT DT
Applying the steady-state approximation for the ES, we have
D;%3=
K;%=;3= K ;%3= K ;%3= (3.5)
DT
Since we cannot measure the free enzyme concentration experimentally;
the equilibrium between the free enzyme concentration and the bound enzyme
concentration is given as,
[E]0 = [E] + [ES] (3.6)
where [E]0 is the total enzyme concentration which is measurable, [E] is free
enzyme concentration and [ES] is the bound enzyme concentration.
From Eq. (3.6), we get
[E] = [E]0 [ES] (3.7)
Substituting Eq. (3.7) in Eq. (3.5) and further simplifying,
K;%= ;3=
;%3= (3.8)
K K K;3=
D;0=
As the [S] becomes very large, the + is commonly referred to as the
DT
maximal velocity of reaction (vmax). Hence, Eq. (3.4) can be written as
D;0=
VMAX K ;%3=
DT
Therefore, Eq. (3.11) can be written as
VMAX ;3=
R (3.12)
+M ;3=
which is a constant.
Case 3. When [S] = Km, Eq. (3.12) becomes
VMAX
R
This means the rate of product formation is half the maximum velocity.
All these can be represented on a graph of r vs [S] as shown in
Figure 3.6.
vmax
vmax
vmax Zero order
2
O [S]
Km = [S]
K K
ZZZZX
% 3 YZZZZZ
Z %3
% 0
K
K
where +M is called the dissociation constant.
K
Now, substituting Eq. (3.13) into Eq. (3.4), we get
K ;% =;3=
R
+M
;3=
VMAX ;3=
or R (3.15)
+M
;3=
which is also called the MichaelisMenten equation.
Note: K¢m is also MichaelisMenten constant. As it is derived assuming
the rapid equilibrium approach, a prime is used over Km.
We have seen that the rate of reaction changes from the first order to
the zero order whenever the first two cases are considered. To explain this we
shall make use of the physical interpretation. Each molecule of an enzyme
has one or several active centres or the sites onto which the substrate
molecule must bind for a reaction to take place.
1. At low substrate concentrations, most of the enzyme active centres
remain unoccupied at any point of time. So the rate of reaction is
proportional to the substrate concentration, which is the first order.
2. As the substrate concentration increases, the active centres are fully
occupied and hence the rate of reaction also increases, which is the
zero order.
Enzymes and Enzyme Kinetics 75
+ MD;3=
D ;3= V MAXDT (3.17)
;3=
D;3=
+M Ô ;3= Ô D;3= Ô
V MAX DT
we get
Km ln[S0/S] + (S0 S) = vmaxt (3.18)
3 3 V MAXT
+M (3.19)
LN33 LN33
Solution
We know that when [S] ? Km, Eq. (3.12) becomes
r = vmax
= k2[E]0 (Q [ES] = [E]0 when [E] = 0)
When Km = [S],
VMAX
R
We know that
K K
+M
K
Therefore,
K K
;3=
K
As k2 = k1,
K
;3=
K
Now substituting values,
K
DMMG
K
Example 3.2 For the enzyme catalysis, the following mechanism is shown
with usual notations. Using the steady-state approximation for the complex
ES, derive the rate equation for the formation of the product during the
initial stages.
K K
% 3
%3 %0
K K
Solution
Using the SSA, write the equation for d[ES]/dt and equate it to 0.
Further, use the enzyme conservation equation and eliminate for concentra-
tion of E. Substitute for [E] in d[ES]/dt = 0. Further, simplify to get the
value of [ES].
The rate of product formation in the initial stages can be written as
D;3= D;0=
R K ;%3= K ;%=;0=
DT DT
Put [P] = 0 (initial stages). Therefore,
r = k2[ES]
Enzymes and Enzyme Kinetics 77
Example 3.3 The following data have been obtained on the enzyme
catalyzed reaction at various substrate concentrations:
[S], mM 0.4 0.6 1.0 1.5 2.0 3.0 4.0 5.0 10.0
Rate, r
(arbitrary
units) 2.41 3.33 4.78 6.17 7.41 8.70 9.52 11.50 12.50
Solution
As the rate of reaction also depends on the concentration of substrates,
we can draw a graph of rate vs substrate concentrations to obtain the profile
shown in Figure 3.7.
vmax
vmax
r 2
Using Eq. (3.15), from the graph find vmax. Then taking [S] = 10.0 at
which r = 12.50, we get Km = 2.0 mM.
Example 3.4 Calculate the ratio of the substrate concentration required for
75% of vmax to the concentration required for 25% of vmax.
Solution
According to the MichaelisMenten expression given in Eq. (3.15), we get
78 Biochemical Engineering: Principles and Concepts
R ;3=
VMAX +M ;3=
or [S]75 = 3 Km
Similarly,
R ;3=
VMAX +M ;3=
or [S]25 = 0.333 Km
The ratio
;3=
;3=
VMAX ;3=
R
+M ;3=
+M ;3=
R VMAX ;3=
+M
(3.20)
R VMAX ;3= VMAX
1/r
slope
m = Km/vmax
Intercept
c = 1/vmax
1/Km 1/[S]
Figure 3.8 Lineweaver–Burk plot.
or R+M (3.21)
R VMAX
;3=
Plot a graph of r vs r/[S] to get the slope and the intercept as shown in
Figure 3.9.
r
vmax
Slope
m = Km
vmax/Km
r/[S]
Figure 3.9 Eadie–Hofstee plot.
80 Biochemical Engineering: Principles and Concepts
VMAX ;3=
R
+M ;3=
;3= +M ;3=
(3.22)
R V MAX V MAX
Plot a graph of [S]/r vs [S], and get the profile as shown in Figure 3.10. This
plot is used to determine vmax more accurately.
[S]/r
m = 1/vmax
Km/vmax
Km [S]
Figure 3.10 Hanes–Woolf plot.
Example 3.5 The following data have been obtained for two different initial
enzyme concentrations for an enzyme-catalyzed reaction.
Solution
Using Eq. 3.22, find [S]/r for both initial concentrations of enzymes.
As [S] is g/L and r is g/Lmin, [S]/r is in minutes.
Tabulate the [S]/r values for both initial concentrations of enzymes.
For [E]0 = 0.015 g/L, plot [S]/r vs [S] values (Figure 3.11).
[S]/r
[S]
Figure 3.11 Example 3.5 (part 1).
Similarly, for [E]0 = 0.00875 g/L, plot [S]/r vs [S] values (Figure 3.12).
[S]/r
Km/vmax = 10 minutes
[S]
Figure 3.12 Example 3.5 (part 2).
82 Biochemical Engineering: Principles and Concepts
= 1.0/0.00875
Example 3.6 The following data on the substrate removal rate and the
substrate concentration is available for some waste treatment.
Estimate the value of substrate removal rate constant Km and the value
of vmax.
Solution
From the tabulated values, find 1/r and 1/[S] so that these can be used
in terms of the double reciprocal method or LineweaverBurk method.
The reciprocals are tabulated as under:
From the above values plot 1/r vs 1/[S] and from which determine the
slope and the intercept.
The slope from the graph is
Km/vmax = 0.0366
= 0.0476 min1
Enzymes and Enzyme Kinetics 83
3.5.1 pH Effects
Certain enzymes have ionic groups on their active sites and these ionic
groups must be in a suitable form (acid or base form) for the enzymes to
function. Variations in the pH values of the medium results in changes in the
ionic form of the active site, and changes in the activity of an enzyme and
hence the reaction rates. The changes in the pH values may also alter the
three dimensional shape of an enzyme. For these reasons, enzymes are only
active over a certain pH range. The pH of the medium may affect the
maximum reaction rate vmax, Km and the stability of an enzyme. In some
cases, the substrates may contain ionic groups, and the pH of the medium
affects the affinity of the substrate to the enzyme. Figure 3.13 shows a graph
of per cent maximal activity vs pH.
100
B (cholinesterase)
Per cent maximal activity
A (trypsin)
0
pH 12.0
Figure 3.13 Per cent maximal activity vs pH.
The profiles shown in the figure are in particular for two different
enzymes:
A: Approximate activity for trypsin
B: Approximate activity for cholinesterase
Consider the following scheme:
E + H+
ZZZX
YZZZ
K2
+M
¢ K
ZZZZZ
X
%( 3 YZZZZ
Z %(3
%( 0
+
H+
ZZZX
YZZZ
K1
EH+2
84 Biochemical Engineering: Principles and Concepts
;%(=;3=
+M (3.23)
;%(3=
;%(=;( =
+ (3.24)
;%( =
;% =;( =
+ (3.25)
;%(=
r = v = k2 [EHS] (3.26)
The enzyme conservation equation is given by
[E]0 = [E] + [EH] + [EH+2 ] + [EHS] (3.27)
Substituting Eqs. (3.24) and (3.25) in the above equation,
+ ;%(= ;%(=;( =
;%= ;%(= ;%(3=
;( = ;+ =
È ;( = ;+ = Ø
or ;%= ;%(= É Ù ;%(3=
Ê + ;( =Ú
;%= ;%(3=
or ;%(= (3.28)
È ;( = + Ø
É + Ù
Ê ;( =Ú
È ;( = + Ø
or +M É Ù ;%(3= ;%(3=;3= ;%= ;3=
Ê + ;( =Ú
;%= ;3=
;%(3= (3.29)
È ( + Ø
+M É Ù ;3=
Ê + ( Ú
Enzymes and Enzyme Kinetics 85
K ;%= ;3=
R
È ;( = + Ø
+M É Ù ;3=
Ê + ;( =Ú
VMAX ;3=
or R (3.31)
+M APP ;3=
where
È ;( = + Ø
APP
+M +M É Ù
Ê + ;( =Ú
As a result of this, the pH optimum of the enzyme is between pK1 and
pK2. In general, increase in pH considerably influences the enzyme activity.
Each enzyme has an optimum pH at which the velocity is maximum. Below
and above this pH, the enzyme activity is much lower and at the extreme
pH, the enzyme is totally inactive. Most of the enzymes (higher organisms)
show the optimum activity at a pH of 6.08.0. But there are some like pepsin
that show optimum activity at pH 12, acid phosphatase at pH 45, and
alkaline phosphatase at pH 1011. In reality, the theoretical prediction of
the optimum pH of enzymes requires knowledge of the active site
characteristics of enzymes which is very difficult.
100 Temperature
activation
%
max I II
activity Thermal
denaturation
0
10 Temperature (°C) 60 50
Figure 3.14 Effect of temperature on enzyme activity.
86 Biochemical Engineering: Principles and Concepts
3.5.4 Activators
Metals act as activators of enzyme velocity through various mechanisms.
They combine with the substrate to form a temporary Metal-Enzyme-
Substrate (MES) complex, directly participate in the reaction and bring
about a conformational change in the enzyme. For example, inorganic
Enzymes and Enzyme Kinetics 87
metallic ions basically cations like, Mg2+, Mn2+, Zn2+, Ca2+, Co2+ etc. activate
the enzymes and rarely anions like Cl activate the enzymes like amylases.
3.5.6 Time
Always under ideal conditions, the time required for the enzyme reaction is less.
3.5.7 Radiations
Certain enzymes become inactive due to exposure to UV, beta, gamma and
X-rays. The active sites are lost due to the loss or damage of the tertiary
structure of the enzyme molecule.
KI
ZZ
ZZ
X
EI
vmax
+I
Vel.
Km K¢m [S]
Figure 3.15 Enzyme velocities vs concentration of substrates.
1/r +I
I >0
I =0
Km/vmax
1/vmax
;%=;3=
+M (3.33)
;%3=
;%=;)=
+) (3.34)
;%)=
;%=;)=
[E]0 = [E] + [ES] + (3.36)
+)
or [E]0 = [E]
;)= + [ES]
+)
;%= ;%3=
or ;%= (3.37)
;) = + )
;%= ;3=
or ;%3= (3.38)
È ;)= Ø
;3= +M É
Ê + ) ÙÚ
;%= ;3=
R K
È ;)= Ø
;3= +M É
Ê + ) ÙÚ
VMAX ;3=
R
;3= +M APP
È ;)= Ø
where +M APP +M É Ù
Ê +) Ú
The net effect of competitive inhibition is an increased value of
K¢m,app and therefore reduced reaction rate. This type of inhibition can be
overcome by high concentrations of substrate.
Reversible non-competitive inhibition: The inhibitor I binds at a site other
than the active site on the E surface. This impairs the enzyme function. The
inhibitor has no structural resemblance with the substrate. However, there
exists usually a strong affinity for I to bind at the second site. There is no
interference with E-S binding, but catalysis is prevented possibly due to
distortion in enzyme conformation.
I generally binds with E as well as ES complex. The mechanism of
reversible non-competitive inhibition is given as follows:
ZZZZZ
% 3 YZZZZX
Z %3 %0
+M K
+
+
I I
ZZZX
Z
ZZZX
Z
YZZ
YZZ
KI KI
ZZZZZ
X
%) 3 YZZZZ
Z %3)
+M
Enzymes and Enzyme Kinetics 91
;%=;)= ;%3=;)=
+) (3.40)
;%)= ;%3)=
vmax
Vel.
vmax
+I
vmax/2
Km [S]
Figure 3.17 Enzyme velocities vs substrate concentration.
1/r
+I
I >0
I =0
1/vmax
1/vmax
1/K¢m 1/[S]
Figure 3.18 Lineweaver–Burk plots for reversible non-competitive inhibition.
;%=;)= ;%3=;)=
or ;%)= ;%3)= (3.41)
+) +)
r = k2[ES]
We know that
[E]0 = [E] + [ES] + [EI] + [ESI] (3.42)
92 Biochemical Engineering: Principles and Concepts
;%=;)= ;%3=;)=
;%= ;%= ;%3= (3.43)
+) +)
È ;)=Ø È ;)=Ø
or ;%= ;%= É Ù ;%3= É Ù
Ê +) Ú Ê +) Ú
È ;)=Ø È ;)=Ø
or ;%= É Ù ;%= ;%3= É Ù
Ê +) Ú Ê +) Ú
È ;)=Ø
;%= ;%3= É Ù
or Ê +) Ú (3.44)
;%=
È ;)=Ø
É + Ù
Ê ) Ú
Î È ;)=Ø Þ
Ñ;%= ;%3= É Ù ;3=Ñ
Ñ Ê +) Ú Ñ
+M Ï ß
Ñ È ;)=Ø Ñ
Ñ É + Ù ;%3= Ñ
Ð Ê ) Ú à
or
È
+M É
;)= Ø ^
;%= ;%3= ;)=+ ) ;3= `
Ù
Ê +) Ú ;%3=
;%= ;3=
;%3=
;)=+ ) ;3=
;%= ;3=
or ;%3=
È ;)= Ø
É + Ù +M ;3=
Ê ) Ú
Enzymes and Enzyme Kinetics 93
VMAXAPP
R (3.44b)
+M
;3=
where
VMAX
VMAXAPP
;)=
+)
The net effect of non-competitive inhibition is a reduction in vmax. High
substrate concentrations would not overcome this type of inhibition. Extra
reagents need to be added to block the binding of the inhibitor to the
enzyme.
Reversible uncompetitive inhibition: It is an uncommon kind of reversible
inhibition. The inhibitor I does not bind with E but only binds with ES
complex. The mechanism of reversible uncompetitive inhibition is shown as
follows:
M X
ZZZZZ
+ K
% 3 YZZZZZ %3 0
+
I
ZZZX
YZZZ
KI
ESI
Now refer to Figures 3.19 and 3.20. From these we observe that:
1. Km value decreases
2. vmax value decreases
94 Biochemical Engineering: Principles and Concepts
vmax
+I
V
[S]
Figure 3.19 Effect of I on enzyme velocity.
1/r
I>0
I =0
1/vmax
1/vmax
–1/Km –1/Km 1/[S]
Figure 3.20 Lineweaver–Burk plots for reversible uncompetitive inhibition.
;%=;3=
From the mechanism shown above, we get K¢m =
;%3=
;%3=;)=
+)
;%3)=
;%3=;)=
or ;%3)= (3.45)
+)
The enzyme conservation equation is given by
;%3=;)=
[E]0 = [E] + [ES] +
+)
Enzymes and Enzyme Kinetics 95
È ;)=Ø
or ;%= ;%= ;%3= É Ù
Ê +) Ú
È ;)= Ø
or ;%= ;%= ;%3= É Ù (3.47)
Ê +) Ú
We have
;%=;3=
+M
;%3=
Now substituting Eq. (3.47) in the above equation,
;%=;3= È ;)= Ø
or +M ;3= É Ù
;%3= Ê +) Ú
;%=;3= È ;)= Ø
or +M
;3= É Ù
;%3= Ê +) Ú
;%= ;3=
or ;%3= (3.48)
È ;)= Ø
+ M ;3= É
Ê + ) ÙÚ
We know that
r = k2 [ES]
Now, substituting Eq. (3.48) in the above equation, we get
K ;%= ;3=
R
È ;)= Ø
+M
;3= É
Ê + ) ÙÚ
VMAX ;3=
or R
È ;)= Ø
+M
;3= É Ù
Ê + )Ú
96 Biochemical Engineering: Principles and Concepts
+M X
ZZZZZ % 0 K
% 3 YZZZZ Z %3
+
S
X
ZZ
ZZ
KSI
ZY
Z
ES2
Refer to Figure 3.21 (the LineweaverBurk plot). It is no longer linear.
However, towards the 1/vmax axis, which is the high substrate end, the line
curves upwards to show the reduction in velocity.
1/r
1/vmax
–1/Km 1/[S]
Figure 3.21 Profiles for the substrate inhibition (1/r vs 1/[S]).
Reaction
v rate with the
same vmax
and Km
Substrate
Inhibition
[S]max [S]
Figure 3.22 Comparison of substrate inhibition and pure enzymatic reactions.
From the mechanism shown above, we can define the following equations:
;%=;3=
+M
(3.50)
;%3=
98 Biochemical Engineering: Principles and Concepts
;%3=;3=
+ 3) (3.51)
;%3 =
;%3=;3=
or ;%3 = (3.52)
;+3) =
and
r = k2[ES]
The enzyme conservation equation is given by
[E]0 = [E] + [ES] + [ES2]
Substituting Eq. (3.52) in the above equation, we get
;% 3 =;3 =
[E]0 = [E] + [ES] +
+ 3)
È ;3= Ø
or ;%= ;%= ;%3= É Ù
Ê + 3) Ú
È ;3= Ø
or ;%= ;%= ;%3= É Ù (3.53)
Ê + 3) Ú
ÑÎ È ;3= Ø ÑÞ
Ï;%= ;%3= É ;3=ß
;%=;3= Ê + 3) ÙÚ Ñ
Use +M ÐÑ à
;%3= ;%3=
È ;3= Ø
;%= ;3= ;%3=;3= É
Ê + 3) ÙÚ
;%3=
;%=;3= È ;3= Ø
or +M
;3= É Ù
;%3= Ê +3) Ú
;%=;3=
or ;%3=
È ;3= Ø
+M
;3= É
Ê + 3) ÙÚ
Enzymes and Enzyme Kinetics 99
;%=;3=
or ;%3=
;3=
+M
;3=
+ 3)
Substituting the above equation in, r = k2 [ES], we get
K ;%= ;3=
R
;3=
+M
;3=
+ 3)
VMAX ;3=
R
;3=
or (3.54a)
+M
;3=
+ 3)
VMAX
or R (3.54b)
+M ;3=
;3= +3)
;3=
+ 3)
4CVG
/ / +PETGCUKPI
EWTXG KPJKDKVQT
+PETGCUKPI
CEVKXCVQT
%QPEGPVTCVKQPQHUWDUVTCVG
From Figure 3.23, it is apparent that all the lines are heading towards the
same Vmax value which means that the effector has functioned by changing
the substrate binding which will be seen as a change in the Km value. Such
systems are called K-systems while some enzymes have effectors which
function by altering the Vmax values and are called V-systems.
Some enzymes like regulatory enzymes have more than one substrate
binding site. The binding of one substrate to the enzyme facilitates binding
of other substrate molecules. This behaviour is called allostery (allo means
other or different) or cooperative binding.
The rate expression of allosteric inbibition is as follows:
D;3= VMAX ;3=N
R (3.55)
DT ;3=N
+M
where n = cooperative coefficient. If n > 1, it signifies positive cooperativity.
The plot of r vs [S] given in Figure 3.24 shows the comparison between
the MichaelisMenten (MM) and allosteric kinetics.
r
MichaelisMenten
Allosteric
[S]
Figure 3.24 Comparison between Michaelis–Menten and allosteric kinetics.
102 Biochemical Engineering: Principles and Concepts
The Dixon plot to find the Inhibitor constant: The Dixon plot is a
convenient method of calculating the inhibitor constant KI. It has certain
constraints as well, as it cannot be used to calculate the Km, vmax etc.
The reaction velocity is measured at a fixed concentration of substrate
but at varied inhibitor concentrations. Figure 3.25 shows the reciprocal of
velocity against inhibitor concentration values. Indeed the plot is shown for
three different substrate concentrations for non-competitive inhibition
process.
Enzymes and Enzyme Kinetics 103
I/velocity
KI
Concentration of inhibitor
We find here a vertical line dropped from the point where the lines are
intersecting down to the inhibitor axis finally giving KI. For a classical non-
competitive inhibitor, the intersection would be on the axis so the constant
is read off directly. For competitive or mixed inhibition, the intersection will
be some way above the axis.
While for uncompetitive inhibition, the lines would be parallel and there
is no intersection as KI is irrelevant to these inhibitors.
[E]act = [E]act, t
T D;%=ACT T
Ô ;%=ACT KDE Ô DT
LN;%=ACTT
or KDE T
;%=ACT
%ACT % )
Assume that the deactivation process is slow and that free E will
deactivate faster than the enzyme in the bound state, i.e. ES complex.
We can write:
D;%=ACT
;%=KDE (3.58)
DT
We know that
[E]0 = [E] + [ES]
Replacing [E]0 with [E]act,
[E]act = [E] + [ES]
or [E] = [E]act [ES] (3.59)
Enzymes and Enzyme Kinetics 105
D;%=ACT
KDE ;%=ACT ;%3= (3.60)
DT
;%= ;3=
;%3= (3.61)
+M ;3=
D;%=ACT È ;%=;3= Ø
KDE É;%=ACT Ù
DT Ê +M ;3=Ú
D;%=ACT È ;%=ACT;3= Ø
KDE É ;%=ACT Ù
DT Ê +M ;3=Ú
È ;3= Ø
KDE ;%=ACT É Ù
Ê +M ;3=Ú
È + M ;3= ;3= Ø
KDE ;%=ACT É Ù
Ê + M ;3= Ú
È +
KDE ;%=ACT É M ØÙ
Ê +M ;3=Ú
È Ø
D;%=ACT É Ù
KDE ;%=ACT É Ù
DT É ;3= Ù (3.62)
ÉÊ + ÙÚ
M
;%=ACT ? K T
;%=ACT E DE
KDE T
or E
LN
or T T
KDE KDE
The above expression relates the deactivation constant and the time
factor.
and
D;3 = K ;%= ;3 =+
R (3.68)
DT ;3 =+ ;3 =+
where [E]0 = [E] + [ES], the total enzyme concentration.
The above equations indicate that if two reactions are catalyzed by the
same enzyme, the individual velocities are slower in the presence of both
substrates than in the absence of one of the two substrates.
Let us say that if [STotal] is the total concentration of substrates present
in the system i.e. [STotal] = [S1] + [S2] (3.69)
Then the overall rate of disappearance of the total substrates is given as
D ;34OTAL = È D ;3 = D ;3 =Ø
R4OTAL É
DT Ê DT DT ÙÚ
S1 S2 P1 P2
We find that the central complex is identical in both the cases while the
subsequent step shown below is same with an ordered mechanism.
(ES1S2) (EP1P2)
From the above, we have now a choice of sequence of product release as
shown below:
(EP1P2) EP2 + P1
EP2 E + P2
or
(EP1P2) EP1+ P2
EP1 E + P1
The Cleland approach for the above is as shown (Figure 3.27).
S1 S2 P1 P2
ES1 EP2
E E
ES2 EP1
(ES1S2) (EP1P2)
S2 S1 P2 P1
S1 P1 S2 P2
Energy link control: This type of control involves the use of energy carriers
that serve as effectors or modulators. Adenylates such as adenosine
triphosphate (ATP), other purine or pyrimidine nucleotides are the effector
molecules.
These are certain enzymes that show sensitivity to the absolute
concentrations of ATP, ADP and even AMP; others seem to react to the
ratio of a given two of these nucleotides.
Certain other compounds are Guanosine triphosphate (GTP), Uridine
114 Biochemical Engineering: Principles and Concepts
P1, 2, 3, 4 Products
and Intermediates
E1 L-isoleucine
E2 E3 E4 E5
P1 P2 P3 P4
CH3
|
CH2
|
HCCH3
|
HCNH2
|
COOH
E1 E2 E3 E4 E5
A (substrate) B C X Y Z
SUMMARY
In this chapter, we learnt the following:
· Enzymes are protein biocatalysts synthesized by living cells. They are
classified into six major classesoxidoreductases, transferases,
hydrolases, lyases, isomerases and ligases.
· An enzyme is specific in action, possessing active centres, where the
substrate binds to form the enzymesubstrate complex, before the
product is formed.
· Concentration of enzyme, concentration of substrate, temperature,
pH, etc. are the factors that influence the activity of an enzyme. The
substrate concentration to produce half-maximal activity is called
Michaelis constant.
· The mechanism of enzyme action is explained by lock and key model,
induced fit theory and the substrate strain theory.
· Most of the enzymes require the presence of non-protein substances
called cofactors and coenzymes for their action.
· The kinetic parameters can be evaluated by using LineweaverBurk,
EadieHofstee and HanesWoolf methods.
· Enzyme activities are inhibited by reversible (competitive, non-
competitive and uncompetitive), irreversible and allosteric manner.
· Reversible inhibition is of three types: reversible competitive,
reversible non-competitive and reversible uncompetitive inhibition.
· Substrate and product inhibitions also occur along with irreversible
inhibition and allosteric inhibition.
· Microorganisms have evolved a variety of enzyme regulatory
mechanisms that accommodate and adjust to the changing needs of
a cell in a changing habitat and surroundings.
· The three modes of regulation of enzyme activity are: energy link
control, feedback inhibition and precursor activation.
EXERCISES
3.1 List the six major classes of enzymes as proposed by the Enzyme
Commission, International Union of Biochemists, in the year 1961.
3.2 Define the following terms:
(a) Holoenzyme, (b) Apoenzyme, (c) Coenzyme, (d) Cofactors,
(e) Monomeric and Oligomeric enzymes, and (f) Multienzyme
complexes.
3.3 Brief the following:
(a) Proximity effect, (b) Orientation effect, (c) Isozymes and
Ribozymes, and (d) Turn over number.
Enzymes and Enzyme Kinetics 117
3.17 The following data have been obtained for two different initial
enzymes concentrations for an enzyme-catalyzed reaction.
Find the following: (a) Km (b) vmax for [E]0 = 0.021 g/L (c) vmax for
[E]0 = 0.00935 g/L and (d) k2 by using HanesWolf method of
transformation.
3.18 During the hydrolysis of urea, which shows inhibition, the following
data is obtained.
% 0
K K K
ZZZZ
% 3 YZZZ
X ZZZZ
Z ;%3= YZZZ
X
Z ;%3=
K K
K
ZZZZX
% 3 YZZZZZ
Z ;%3=
K
K
ZZZZX
%3 3 YZZZZZ
Z ;%33=
K
K
;%33= ;%3= 0
K
;%3=
% 0
Derive a rate expression for product formation assuming quasi state
for [ES] and [ESS].
Hint: Consider d[ES]/dt = 0; d[ESS]/dt = 0
Plot r vs [S] and calculate the value of Km and the limiting rate.
3.24 Michaelis-Menten constant of an enzyme hexokinase for glucose
substrate is 0.15 mM and for fructose substrate is 1.5 mM.
Assuming that vmax is same for both the substrates, calculate the
values for the rate as vmax per cent for each substrate, when [S] = 0.15
mM, 1.5 mM and 15 mM. Further, show that which substrate does
hexokinase show a greater affinity for?
Hint: According to the Michaelis-Menten equation as per 3.12 or
3.15, we have
V MAX ;3=
R
+ M ;3=
It is given that vmax is same for the substrates and Km = 0.15 mM
for glucose and Km = 1.5 mM for fructose.
For the concentration of substrates as given in the problem,
[S] = 0.15 mM, for glucose and fructose, we can find out the values
for respective substrates as
For glucose, at [S] = 0.15 mM, the value of r or v is 50% vmax, since
[S] = Km.
Similarly, for fructose, at the above substrate concentration, the
value of r or v is 9.1% vmax.
In the same way, we can find out the values for other concentrations
of substrate.
For [S] = 1.5 mM, for glucose, r or v is 91% vmax; for fructose it is
50% vmax, since [S] = Km.
For [S] = 15 mM, for glucose, it is 99% vmax; and for fructose it is 91%
vmax.
From the above estimations, it should be clear that hexokinase has
more affinity towards glucose than fructose.
3.25 The following data is obtained in an enzyme catalyzed reaction.
Using the same determine the values for Michaelis-Menten constant
Km, and the maximum velocity of the reaction.
Data:
S,
mol/lit. 4.1 ´ 103 9.5 ´ 104 5.2 ´ 104 1.03 ´ 104 4.96 ´ 105 1.05 ´ 105
r ´ 106
mol/lit. 177 173 125 106 80 67
Enzymes and Enzyme Kinetics 121
Hint: Using the data given above, determine the reciprocal values
of S and r for each value given i.e. find out the values of 1/S and
1/r to get the linear profile on the ordinates of 1/r vs. 1/S.
Next, plot all the values of 1/r vs. 1/S to get a straight line.
Make use of Lineweaver-Burk method as shown in Figure 3.25.
1/r
Slope = Km/vmax
Intercept = 1/vmax
1/S
Figure Exercise 3.25.
From the graph, find out 1/vmax and the slope of the line is Km/vmax.
Then finally find out Km and vmax.
3.26 An antibiotic, Penicillin is hydrolyzed and thereby rendered inactive
by an enzyme Penicillinase, usually present in the resistant bacteria,
Staphylococcus aurens.
The mass of this enzyme measured is 29.6 k Daltons. The amount of
penicillin hydrolyzed in 1 minute in a 10 ml solution containing
109 grams of purified Penicillinase was measured as a function of
concentration of Penicillin.
Assume that the concentration of Penicillin doesnt change
appreciably during the assay. Using the data given below,
Data:
Penicillin
´ 105 M 0.1 0.3 0.5 1.0 3.0 5.0
Amount hydrolyzed,
moles ´ 109 0.11 0.25 0.34 0.45 0.58 0.61
1/r
Slope = Km/vmax
Intercept = 1/vmax
1/S
Figure Exercise 3.26
Find Km and vmax from the graph. The values obtained are the
solution for (i).
In the second part of the problem, to find the TON:
Molecular weight = 29.6 grams/gram-mole = 1Dalton.
29.6 grams implies 1 gram-mole
1 gram implies gram-mole.
Amount of Penicillinase present = 1 ´ 109 grams. (Given)
Now converting into gram-moles implies
ÎÑ ÞÑ
(1 ´ 109) Ï ß
ÑÐ Ñà
The concentration of Penicillinase, an enzyme is [E] = 0.03378 ´ 1012
moles/m3.
Using the relation:
vmax = k2[E0]
k2 = vmax/[E0]
Here k2 is the TON. Substitute the values of vmax and [E0] and find k2.
Enzymes and Enzyme Kinetics 123
S, mol/lit: 3.3 ´ 10-4 5.0 ´ 10-4 6.7 ´ 10-4 1.65 ´ 10-3 2.21 ´ 10-3
r, mol/lit-min:
No Inhibitor- 56 71 88 129 149
With Inhibitor- 37 47 61 103 125
with I
1/r
I = 0
1/vmax
From Figure 3.27, calculate Km, vmax and KI for both competitive
and non-competitive inhibition, although the inhibition is
competitive.
(iii) To find the pesticide or inhibitor concentration, [I]:
Enzyme solution is 50 ml
Substrate solution is 50 ml and sample is 25 ml. (Given)
Total solution is 125 ml.
Å Å n
The concentration of substrate is = 6.4 ´ 10-6 M/ml. = [S]
Finally, using the equation for competitive inhibition, find [I].
124 Biochemical Engineering: Principles and Concepts
3.28 For the initial substrate concentration, the initial rate of reaction
with and without inhibitor is shown below.
Data:
Substrate, mm/lit: 6.7 3.5 1.7 0.3
Initial reaction rate,
mm/min.
I = 0 0.30 0.25 0.16
I = 146 mm 0.11 0.08 0.06
Identify which type of inhibition holds good for the above and also
find all the rate constants.
Hint: From the above data, write the reciprocals for [S] and r for both
without inhibitor and with inhibitor, i.e. 1/[S] and 1/r respectively.
Plot the graph 1/[S] vs. 1/r using the above values.
Based on the nature of graph, predict the type of inhibition.
Using the intercept, slope values find out the values for Km, KI, and
vmax, etc.
3.29 In the study of enzyme kinetics, the following results were obtained.
Evaluate Km using (a) the r vs [S] plot and (b) the LineweaverBurk
plot.
Chapter 4
Industrial Enzymes and
Applications
All enzymes are derived from three sourcesanimal, plant and microbial
sources. Animal and plant sources have been found to be relatively expensive,
for example rennin which is derived from calfs stomach and papain derived
from unripe papaya. On the contrary, the enzymes derived from microbial
sources are comparatively cheap and the process can be scaled up easily. As
microbes have rapid doubling time, these are exploited both commercially
and industrially to meet the current market demands. Only those microbes
or microorganisms certified as safe are used to synthesize enzymes parti-
cularly to be used in food and pharmaceutical industry.
In this chapter we shall survey some of the enzymes which are utilized
in the absence of life. Such biological catalysts include extracellular enzymes
secreted by cells in order to degrade polymeric nutrients into molecules small
enough to permeate cell walls. Applications of various enzymes like
hydrolytic, proteolytic, non-hydrolytic, etc. are discussed. In addition to these,
the enzyme market and the commercial production of enzymes are also
included.
Recombinant DNA Technology also known as Genetic Engineering is a
much heard concept in the present era. This is also utilized in synthesizing
newer enzymes from the microbial sources in order to meet the current
market demands.
Certain applications demand the use of a pure extract, for example an
enzyme, glucose oxidase, which is required in the desugaring of eggs, should
be free of any protein splitting enzymes so that the protein part of an egg
is essentially not destroyed. In the same way, the proteolytic enzymes that
are used in the process of meat tenderization, to be injected before
slaughtering, should be free of any compound which can cause serious
physiological changes. Undoubtedly, these enzymes are also used in the
clinical and medical diagnosis.
Basically, enzymes are intracellular and extracellular. Intracellular
enzymes are present in cells. In order to synthesize, the cells require grinding,
125
126 Biochemical Engineering: Principles and Concepts
mashing, lysing or otherwise killing and splitting whole cells that open frees
the intracellular enzymes. While extracellular enzymes, which are utilized in
the absence of life, degrade the polymeric nutrients into small monomers
which can permeate the cell walls.
Trypsin
Chymotrypsinogen Chymotrypsin
Trypsin
Procarboxypeptidase Carboxypeptidase
128 Biochemical Engineering: Principles and Concepts
The products made in enzyme processes are worth billions of dollars. For
example, proteases which hydrolyze proteins to peptides constitute a large
group (about 60%) of enzyme market.
Industrial proteases are obtained from bacteria (Bacillus), moulds
(Aspergillus, Rhizopus and Mico), animal pancreas and plants.
conditions differ from microbe to microbe and even in the same species of a
microbe. There is every chance of foaming taking place inside the fermenter
during the process. To control this, certain amount of oil is added.
After a period of 30 to 120 hours of incubation, extracellular enzymes
are produced by the inoculated microbe in the culture medium. It is seen that
most of the enzymes are produced actually when the exponential growth
phase is complete, but in certain cases the enzymes are produced during the
exponential phase.
During fermentation process, it is also observed that metabolites around
1015% are getting produced besides the desired enzymes as products and for
which the first step mentioned above is carried out carefully to see that the
amount of metabolites formed is less compared to the amount of enzymes.
Metabolites produced are removed by adopting the purification steps.
Once the fermentation process is complete, the broth is retained at 5°C
to avoid contamination. Later the recovery steps are adopted to get the
enzymes. It is also a fact that the recovery of enzymes from a bacterial broth
is more difficult than the filamentous broth as fungal broth can be directly
filtered or centrifuged after pH adjustment.
Therefore, the bacterial broth is first treated with calcium salts to
precipitate calcium phosphate that aids in separation of bacterial cells and
colloids. Subsequent to this, it is then filtered and centrifuged to remove the
cell debris and recover the enzymes. This is followed by the purification step
as discussed below.
Bacterial origin:
Bacillus cereus Penicillinase
B. coagulans a-amylase
B. licheniformis Protease and a-amylase
E. coli Penicillin acylase and b-galactosidase
Klebsiella pneumoniae Pullulanase
Actinomycetes:
Actinoplanes sp. Glucose isomerase
Fungal origin:
Aspergillus niger Amylases, proteases, pectinase, etc.
A. oryzae Lipases, amylases, etc.
Candida lipolytica Lipase
Trichoderma viride Cellulase
S. cerevisiae Invertase
SUMMARY
In this chapter, we learnt the following:
· Enzymes are classified as hydrolytic, proteolytic and non-hydrolytic.
· Esterases, carbohydrases and phosphatases are hydrolytic enzymes.
· Proteolytic enzymes are divided into exopeptidases and endopepti-
dases.
· Proteolytic enzymes are activated from a precursor and metal ions.
They are also called proteoclastic enzymes, and hydrolyze proteins.
· Enzymes can be intracellular and extracellular and are derived from
three sources: animal, plant and microbial sources.
· Glucose oxidase is a non-hydrolytic enzyme and finds use in current
and developing industrial technology.
· Glucose oxidase is added to remove oxygen which otherwise would
react with proteins, producing dark brown colour and further affects
the flavour and the taste of the product.
· The steps in the commercial production of enzymes are:
(a) Isolation of microorganisms, strain development and preparation
of inoculum.
(b) Formulation of medium and preparation.
Industrial Enzymes and Applications 135
EXERCISES
4.1 Give a detailed classification of enzymes with their applications.
4.2 What are proteolytic enzymes? Write on the methods of their
activation.
4.3 What are hydrolytic enzymes? Give a detailed classification with
respect to substrate on which they act upon and the product released.
4.4 Enlist the applications of non-hydrolytic enzymes.
4.5 Enlist some of the enzymes of industrial importance with respect to
their sources and uses.
4.6 How does glucose oxidase act in the preservation of canned food
products?
4.7 Describe all the steps incorporated in the production of enzymes on
a commercial scale.
Chapter 5
Immobilized-Enzyme Technology
5.2 IMMOBILIZATION
Immobilization refers to the confinement or localization of enzymes, so that
they can be reused continuously. There are certainly some reasons as to why
the process of immobilization is desirable. They are as follows:
1. For processing with isolated enzymes
2. Retaining inside a reactor to avoid the loss and consequent replacement
3. In retaining the activity for longer periods of time rather than the
enzymes in solution.
4. Increases catalytic efficiency for the multi-step conversion
5. To get the large throughput of the substrate.
6. To avoid wastage of enzymes as they are costly and expensive.
M E
Note: The bonding or the attachment with others should be indicated not
into the grove, but outside it. Because the grove indicates the active site of
an enzyme.
The cross-linking of enzyme molecules with each other using agents like
glutaraldehyde, bis-diazobenzidine and 2, 2-disulphonic acid is another way
of enzyme immobilization.
Cross-linking can be achieved in several different ways. Enzymes can be
cross-linked with glutaraldehyde to form an insoluble aggregate, adsorbed
enzymes may be cross-linked, or cross-linking may take place following the
impregnation of porous support material with enzyme solution.
The process may cause significant changes in the active site of enzymes,
and also severe diffusion limitations may result.
(Weaker interaction)
(a) Entrapment in porous hollow fibres
E
E
S
P
P
(d) Microencapsulation
Figure 5.2 Physical methods of immobilization.
142 Biochemical Engineering: Principles and Concepts
VMAX
.$A (5.1)
K, ;3B =
where [Sb] is the concentration of substrate in the bulk liquid given as
g/cm3, and kL is the liquid phase mass transfer coefficient in cm/s.
VMAX
$$A from (5.1)
K, ;3B =
Transport of substrate
Fluid (bulk)
(external film)
Sb Substrate
Pb Product
Catalyst
thickness, 2L
Figure 5.3 Symmetric slab of immobilized enzyme accompanying mass transfer and
chemical reactions.
Far away from the catalyst, the substrate concentration and other
process variables such as pH will have the values characteristic of bulk
reaction mixture.
It is seen that the substrate flowing from the bulk fluid crossing the
stagnant film called boundary layer, diffuses into the permeable catalyst,
being immobilized. Here the substrate is utilized and consumed with the
immobilized permeable catalyst and forms the product.
Concentration gradients develop between the bulk solution far from the
catalyst and the reaction events are occurring at the active sites of
immobilized enzyme molecules.
Immobilized-Enzyme Technology 145
5UUWDUVTCVGEQPECV
VJGUWTHCEG
$WNMUWDUVTCVG
EQPEGPVTCVKQPRTQHKNG
0QPRQTQWU
5D
UQNKFUWRRQTV
UWTHCEG
+OOQDKNK\GF (KNOVJKEMPGUUE
GP\[OGU
Figure 5.4 Concentration profile of substrate in a liquid film around bound enzymes on a
non-porous support.
Equating the above Eqs. (5.2) and (5.3) at steady state, we have
VMAX ;3S =
Js = kL{[Sb] [Ss]} = R (5.4)
+M ;3S =
where kL is liquid phase mass transfer coefficient and vmax is the maximum
reaction rate per unit of external surface area.
In the above equation, the number of parameters necessary to specify the
system can be reduced from existing four (kL, Sb, vmax, and Km) to two
(NDa, K) by using the dimensionless variables as defined below.
;3S = VMAX +M
X . $A AND L
;3B = K, ;3B = ;3B =
Using these forms, the above equation in terms of substrate mass balance
is
Î ;3S = Þ
Ñ VMAX ;3 = Ñ
ÑÎ;3B = ;3S = ÑÞ Ñ Ñ
K, Ï ß Ï
B
ß (5.5)
ÑÐ ;3B = Ñà ;3
Ñ +M S Ñ =
ÑÐ ;3B = Ñà
Immobilized-Enzyme Technology 147
;3S =
VMAX
ÎÑ ;3S =ÞÑ ;3B =
K, Ï ß +M ;3S = (5.6)
ÐÑ ;3B = àÑ
;3B = ;3B =
X X
Therefore, where 0 £ x £ 1.0
. $A L X
The above equation is found to be quadratic for x, manipulating
algebraically, we can write as,
CÈ L Ø
X É Ù (5.8)
Ê C Ú
where b º NDa + k 1
The + sign is used for b greater than 0 and sign used for b less than
0, whereas when b = 0, x = Ök.
Using this value for [Ss]/[Sb], either on the left side or right side of the
equation given below
X X
we can evaluate the observed dimensionless reaction rate
. $A L X
R
V MAX
The influence of mass transfer on the overall reaction process is very well
represented using the effectiveness factor h. This effectiveness factor h is
defined as the ratio of observed reaction rate to the rate without mass
transfer resistance, i.e.
/BSERVEDÅREACTIONÅRATE
I
2ATEÅWHICHÅWOULDÅBEÅOBTAINEDÅWITHOUTÅMASSÅTRANSFERÅRESISTANCE
In the above definition for h, the denominator part refers to the fact that
surface concentration of substrate Ss is equal to the bulk concentration Sb i.e.
Ss = Sb.
Therefore,
X
L X
I (5.9)
L
148 Biochemical Engineering: Principles and Concepts
So, that h £ 1.0 and overall, the effect of increasing the mass transfer
resistance is, decrease in the observed activity of catalyst.
X X
For NDa tending to zero, shows that x must approach 1.0
. $A L X
and so for the reaction limited regime NDa ® 0
VMAX ;3B =
I AND R
+M ;3B =
L
I AND R K, ;3B =
. $A
Graphical representations
1. Dimensionless plot of overall reaction rate vs. bulk substrate
concentration (Figure 5.5).
TVOCZ
2TQHKNGUHQTFKHHGTGPV
XCNWGUQH0&C
=5D?
Figure 5.5 Overall reaction rates vs. bulk substrate concentration.
100
h
Diffusion control
[Sb] = 1.0
0.001
NDa 1000
0.1
Figure 5.6 Effective factors vs. Damkohler number.
Graphical approach
The original equation:
V MAX ;3S =
Js = kL{[Sb] [Ss]} = R =
+ M ;3S =
,UQT
VZ
OQNGUEO UGE C
=5U?OQNGURGTNKVTG
Figure 5.7 Flux Js vs. the substrate concentration.
150 Biochemical Engineering: Principles and Concepts
The intersection of the two lines is the reaction rate R that is sustainable.
Further, the responses for two bulk concentrations are also depicted.
When the system is strongly mass transfer limited, [Ss] º 0, since the
reaction is rapid compared to mass transfer and therefore,
r = kL[Sb], for NDa >> 1.0
The above relation shows that the reaction system behaves as a pseudo
first order.
When the system is reaction-limited NDa << 1.0 the rate is expressed as
VMAX ;3B =
R (5.10)
+MAPP ;3B =
where
Î Þ
Ñ VMAX Ñ
+MAPP +M Ï ß (5.11)
ÑÐ ^
K, ;3B = +M ` Ñà
'P\[OGU
KOOQDKNK\GF 4CFKWU4
=5U?
5T 5WDUVTCVG
EQPEGPVTCVKQP
RTQHKNG
T
T4
Figure 5.8 Porous support holding enzymes.
È D ;3= D;3=Ø
$E É (5.12)
Ê DR R DR ÙÚ
D 3 D3 3
G (5.15)
DR R DR 3
C
È VMAX
Ø
É +M Ù
where G 2 É Ù and is called Thiele-modulus. (5.16)
É $E Ù
ÉÊ ÙÚ
D3
With boundary conditions of 3 = 1 at R = 1 and at at R = 0,
DR
Eq. (5.14) can be solved numerically.
Further, stating that the rate of substrate consumption is equal to the
rate of substrate transfer through the external surface of the support matrix
at steady state into the sphere,
We have,
D;3=
RS .S 3 2$E (5.17)
DR R 2
When there are diffusional limitations, the rate per unit volume is
expressed as:
V ;3 =
RS I MAX S (5.18)
+ M ;3S =
where h is effectiveness factor.
Effectiveness factor is defined as the ratio of the reaction rate with
diffusion limitation (or diffusion rate) to the rate without diffusion
limitation.
2EACTIONÅRATEÅWITHÅDIFFUSIONÅLIMITATION
I
2EACTIONÅRATEÅWITHOUTÅDIFFUSIONÅLIMITATION
Immobilized-Enzyme Technology 153
C
I C
G
It is seen that, for a large range of f as 1< f <100 h » 1.0, the reaction
is zero order rate with b tending to zero.
Also for a first order reaction rate, i.e. b tending to infinity h = (h,f)
and h approximated for high values of f then
È Ø
I É Ù (5.19)
G Ê TANH G G Ú
or
È Ø
I É Ù (5.20)
G Ê TANH G G Ú
Based on the above observations, we conclude here that while designing
immobilized enzyme systems using a particular support, the main variables
are vmax and R as substrate concentration [S], Km and De are fixed.
The particle size R should be as small as possible but within the
limitations of particle integrity, resistance to compression and nature of
particle recovery systems.
The maximum reaction rate is found by enzyme activity and
concentration in the support.
High concentration of enzyme will lead to high enzyme activity per unit
of reaction volume, but low h. On the other hand, low concentration of
enzyme will lead to lower enzyme activity per unit volume but has high h.
For maximum conversion rates, particle size should be DP £ 10 mm
(small), and enzyme loading to be optimized.
154 Biochemical Engineering: Principles and Concepts
Attachment: In this, the cells are bound to the surface of a solid support as
shown in Figure 5.12. The attachment relies on the forces of natural adhesion
or be induced by certain chemical means.
The attached cells are in direct contact with the surroundings and hence
subject to the relative motion of particles and fluid when placed in a reactor.
It is therefore likely that some cells will become detached and enter the bulk
fluid phase.
Therefore, this technique is not suitable in the situations where cell free
liquor is required.
Immobilized-Enzyme Technology 159
The systems developed more recently, prefer the use of particulate solid
supports, often of the material such as sand, which when used in a fluidized
bed for instance, provides a greatly increased surface area for attachment per
unit volume of the reactor than in either of the traditional processes.
The art of cell immobilization by attachment is to provide the right
surface in a suitable form for the commercial use and colonized by the desired
organism or population of organisms in as high a density as is possible.
C
D
Figure 5.14 Containment and aggregation.
In aggregation of cells (b), the cells form large aggregates due to the
flocculation of cells and thereby it is possible to retain them continuously in
the operated bioreactors, for example, in the form of packing materials in the
fluidized bed or packed bed reactors.
The artificial flocculants may also be employed to enhance the rate of
aggregation. This is applied in the activated sludge process where the cells
are placed in the form of aggregates.
There are three means of transport across the cell membranes and well
identified. They are:
1. Passive diffusion
2. Facilitated diffusion
3. Active transport
It is, however, realized that the main mechanism that prevails in the
transport across membranes is diffusion. Further, regardless of the mechanism,
the transport characteristics of a given membrane with a given substrate are
often expressed in terms of membrane permeability which is denoted as K.
Membrane permeability K can be computed by considering the
concentrations in and out of the cell as shown below.
It is expressed as
6 È #% # 3 Ø
+ LN É Ù (5.21)
!T ÉÊ # % # ST ÙÚ
1WVUKFGEGNN +PUKFGEGNN
(NQYFKTGEVKQP
4GIKQPQHJKIJ 4GIKQPQHNQY
EQPEGPVTCVKQP EQPEGPVTCVKQP
QHUWDUVTCVGU5 QHUWDUVTCVGU 5
%GNNOGODTCPG
È# Ø
DG0 = RT ln É Ù (5.22)
Ê# Ú
Facilitated diffusion: In this, it is found that the substrates combine with the
carrier molecules to give a facilitated diffusion. Substrates which are on the
outside of cell membrane combine with the carrier molecules present within
the cell forming a complex and diffuse to the other side.
Once having diffused to the other side the complex between the carrier
and substrate splits, discharging the carrier molecule inside the cell.
The same is depicted in Figure 5.16.
1WVUKFGEGNN +PUKFGEGNN
% (NQYFKTGEVKQP
%
4GIKQPQHJKIJ
4GIKQPQHNQY
EQPEGPVTCVKQP
EQPEGPVTCVKQP
QHUWDUVTCVGU
QHUWDUVTCVGU
5 5
%5
%GNNOGODTCPG
1WVUKFGEGNN +PUKFGEGNN
%
4GIKQPQHNQY 4GIKQPQHJKIJ
EQPEGPVTCVKQP EQPEGPVTCVKQP
QHUWDUVTCVGU QHUWDUVTCVGU
5
5
%5
'PGTI[
%GNNOGODTCPG
Fixed bed reactors: These are very common and are convenient to use and
they usually provide the tool for the first test of the applicability of cells
immobilized by certain newer techniques. These are, however, difficult to
scale up and also to quantify Figure 5.18.
Plug flow packed bed reactors operated on a once-through basis may
offer high rates of reaction due to high substrate concentration but are
relatively poor in terms of heat and mass transfer coefficients and this is due
to the low liquid velocities.
The fluidized bed fermenters with recycle show improved mass and heat
transfer characteristics and also improved controllability.
Immobilized-Enzyme Technology 165
2CEMGF
EQNWOPU
CPFHKZGF
DGF
0QPWPKHQTO 2WOR
7PKHQTOUK\G UK\G
.QYRTGUUWTGFTQR%2 *KIJRTGUUWTGFTQR%2
Figure 5.18 Packed bed reactors.
1XGTHNQY
&KUVTKDWVQT .KSWKF
TGE[ENG
#KT
2WOR
Figure 5.19 Fluidized bed reactor.
In this the particles are suspended as a result of the upflow of the fluid
phase and are a focus of much attention these days (Figure 5.19).
166 Biochemical Engineering: Principles and Concepts
Bubble column reactors: These are the reactors which rely on gas
sparging for agitation. Bubble column reactors signify a large height to
diameter ratio. Mixing or agitation is supplied entirely by forcing compressed
gas through spargers into the reactor which then rises through the liquid
(Figure 5.20).
'HHNWGPVICU
5KGXGRNCVGU
+PNGVCKTVJTQWIJ
URCTIGTU
Bubble column reactors have certain advantages over the others. These
are low capital cost, simple mechanical configuration and reduced operating
costs based on the low energy requirements. These are used usually in the
manufacture of beer and vinegar.
These can be used in a batch mode and also in continuous mode
preferably in a counter current fashion.
Note: The other reactor types and configurations are discussed in Chapter 7.
The overall performance of immobilized cell reactors is based on the
following factors:
1. The reaction properties viz. volume of bioreactor, reaction zone
volume, the void volume, the volume occupied by the immobilized
cells plus supports, etc.
2. The fractional particle hold-up
3. The control of biomass hold-up
4. The rate of oxygen supply, and
5. Cell physiology.
Immobilized-Enzyme Technology 167
SUMMARY
In this chapter, we learnt the following:
· Confinement or localization of enzymes so that they can be reused
continuously is called immobilization.
· There are physical methods and chemical methods of immobilization.
· Though immobilization is desirable, there are certain limitations.
· The physical and mechanical properties of supports are important
during process consideration.
· To immobilize enzymes, both natural and artificial supports can be
used.
· Enzyme immobilization is a favoured practice in industry, while
enzymes undergo certain constraints so called diffusional limitations.
· Resistances vary depending upon the nature of the support materials
may be porous or non-porous, hydrodynamic conditions in the vicinity
of support material and the distribution of the enzyme, whether inside
or on the surface of the support material.
· Relative rate of reaction rate and diffusion rate can be taken as an
index of performance for immobilized enzymes over free enzymes.
· Cell immobilization can be a natural process or can be induced by
chemical means or physical means similar to as what we find in the
enzyme immobilizations.
· Techniques for cell immobilization are: attachment, entrapment,
containment and aggregation.
· There are three means of transport across the cell membranes and well
identified. They are:
1. Passive diffusion
2. Facilitated diffusion
3. Active transport.
· The support matrices employed in cell immobilization are usually
mechanically fragile; the bioreactors with low hydrodynamic shear, for
instance the packed bed reactors, the fluidized bed reactors or air lift
reactors are preferred.
· The method of immobilization, characteristics of the particles viz.
size, shape, density, etc. the nature of substrate, the effects of
inhibitors and as well the hydrodynamic and economic factors are the
factors to be considered in selection of bioreactors for immobilized cell
systems.
168 Biochemical Engineering: Principles and Concepts
EXERCISES
5.1 What is enzyme stabilization? Discuss the various strategies for the
same.
5.2 Why is enzyme stabilization required?
5.3 Immobilization of enzymes is desirable. Why?
5.4 Describe the process of adsorption immobilization of enzymes.
5.5 Enlist the chemical methods of enzyme immobilization and further
with neat sketches explain the mechanism of each.
5.6 Explain physical methods of immobilization of enzymes.
5.7 Write a short note on the following:
(a) Interactions and carriers in adsorption method of immobilization
(b) Natural and synthetic supports for covalent enzyme attachment
(c) Microencapsulation
(d) Limitations of immobilization
5.8 Discuss the diffusional mechanisms and limitations observed in the
immobilized enzyme systems.
5.9 Brief on the electrostatic and position effects in the immobilized
enzyme systems.
5.10 Write on the immobilized cell systems and their reactor
configurations.
5.11 Describe the transport mechanisms across cell membranes with their
importance.
Chapter 6
Biomass Production in
Cell Cultures
When a small amount of living cells is added to a liquid solution having
essential nutrients at suitable temperature and pH, the cells will grow. The
growth processes have two different manifestations according to the
morphology of the cells involved. For unicellular organisms which divide as
they grow, increase in biomass (mass of living matter) is accompanied by
increase in the number of cells present. Associated with cell growth are two
other processes: uptake of some material from the cells environment and
release of metabolic end products into the surroundings.
For microbes, growth is their most essential response to their physio-
chemical environment. Growth is a result of both replication and also
changes in cell size. Microorganisms can grow under a variety of physical,
chemical and nutritional conditions.
In this chapter, we shall focus on cell growth, kinetic aspects of cell
growth and situations that apply to other aspects of chemical reactions in
cellular processes. Various reactor configurations with mass balance, with
and without recycle, determination of kinetic parameters and quantification
of growth kinetics are also treated and an insight into the introduction to the
fed batch reactors is included at the end.
169
170 Biochemical Engineering: Principles and Concepts
is used for energy production and some other part is used for biosynthesis and
product formation.
Industrial manufacturing processes that use microorganisms generally
use organic compounds as a source of energy and source of carbon, for
example, carbohydrates.
During the course of growth, carbohydrates are utilized by micro-
organisms and assimilated into the cellular materials of microorganisms
because of which growth is seen. Growth is both in terms of size and numbers.
We say, in general, increase in biomass (mass of living matter).
Growth features vary considerably from organism to organism. Some grow
in numbers (population) and some grow in size. For example, moulds exhibit
growth in terms of length, and mycelia increases as the organism grows. The
growing mould thus increases in size and density and not in numbers.
Even growth requirements may differ. We know that principal elements
associated with microorganisms are C, H, O, N, S and P, and the trace
elements associated are Na, Ca, Fe and Co, etc. Some microorganisms require
N in the form of urea and some require N in the form of ammonium ions.
If carbohydrates are organic constituents in culture as nutrients,
metabolic waste products are CO2 and H2O with liberation of energy.
MICROORGANISMS
Carbohydrates CO2 + H2O + Energy (Release)
(Proceeds in parallel with synthesis reaction)
Unstructured Structured
Actual case
idealized case, i.e. unsegregated and unstructured from the actual case which
was segregated and structured.
Balanced growth: Balanced growth is one of the approximations used. It is
defined as that all cellular synthesis activities are coordinated in such a way
that the average cellular composition is not affected by proliferation of the
population.
Average cell approximation: In this single component, multicomponent
description of cell-to-cell heterogeneity is considered to have a very low
influence on kinetic process of interest. The average cell approximation leads
to an unsegregated perspective.
Since no liquid is added to or removed from the reactor and gas stripping
of culture liquid is low then, VR is constant.
Therefore,
D
# RFI (6.2)
DT I
From Eq. (6.2), rate of change in concentration of i is the rate of
formation, i.e. measurement of rate of change of concentration of i allows the
direct measurement of overall rate of i formation due to reaction. rfi depends
upon the state of cell population, (morphology, composition and age
distribution) and also on the growth medium parameters.
Valve
Flow Pump
meter Sampling points
Air
Culture vessel
Nutrients and
air Product receiver
Xf
X X
Sf
S S
C if Feed, F Feed, F
Ci
V
CSTR above the limiting concentration, we can analyze cell kinetics of the
system separately. Similarly, the same logic holds good for heat transfer
problems during the microbial growth.
Logic 2. Temperature controllers: During growth, temperature certainly
rises. The system is maintained with adequate heat removal capacity and is
equipped with a satisfactory temperature controller. We can assume that it
is an isothermal operation at the desired temperature.
Material balance: Considering the steady state, where all concentrations
within the reactor are independent of time, we can use material balance.
(Rate of addition to reactor) (Rate of removal from reactor) + (Rate
of formation within the reactor) = 0
Mathematically (Figure 6.2),
& # I # IF
or rfi = (6.5)
62
Defining the dilution rate as
D = F/VR,
Eq. (6.5) becomes,
rfi = D[Ci Cif ] (6.6)
The dilution rate D, characterizes the holding time or processing rate of
a CSTR. It is equal to the number of tank liquid volumes which pass through
the vessel per unit time. D is the reciprocal of mean holding or mean
residence time used in biochemical processing. It has the units of per time.
Comparison between batch reactor and CSTR: For a batch reactor, we have
D# I
rfi = (Eq. 6.2)
DT
and for a CSTR we have
rfi = D (Ci Cif) (Eq. 6.6)
In a CSTR, the kinetics determinations are straighter, since we need not
measure the time dependence of concentration.
In a batch reactor, time factor is considered. In a CSTR, cell population
adjusts to a steady environment and achieves almost a state of balanced
growth.
Batch processes are done in a flask at a time using incubators, shakers,
etc. CSTRs are more expensive and complicated. In a CSTR the steady state
is achieved in hours or days, thus magnifies the problem of contamination.
Biomass Production in Cell Cultures 177
& È & Ø
8F É NÙ 8 (6.8)
62 Ê62 Ú
D × Xf = (D µ) X (6.9)
where dilution rate, D = F/VR
As sterile nutrient is given to the liquid feed culture, so Xf = 0. Now
Eq. (6.9) becomes,
DX = µX
or
D = µ (6.10)
It means that the dilution rate is equal to specific growth rate which
shows that a non-zero cell population is revealed and maintained. When
Eq. (6.10) is satisfied, it seems that Eq. (6.9) does not determine X when the
feed is sterile.
The experiments with continuous culture of Bacillus linens confirm the
indeterminate nature of the population level. After a steady state, continuous
operation is achieved at a 6-hour point, and two subsequent interruptions of
the culture are imposed. The behaviour is shown in Figure 6.3.
y x-time (hours)
y-absorbance
temp 26°C
dilution rate = 0.417/hr
6 x
Figure 6.3 Absorbance vs time.
178 Biochemical Engineering: Principles and Concepts
mmax
mmax
m 2
S = Ks
S
Figure 6.4 Substrate concentration vs specific growth rate.
Note: The microorganism requires several substrates for its growth but
it is assumed that all but one is present in excess of requirements and the
substance to which S relates is the limiting substrate component.
With reference to the above graph, the specific growth rate increases with
the increase in concentration of the substrate and reaches a limiting value of
µmax at high substrate levels.
In Eq. (6.11) when S ? Ks, then Ks is small and ignorable.
Therefore, we get
µ = µmax
When S = Ks, Eq. (6.11) becomes
NMAX
N (6.12)
Biomass Production in Cell Cultures 179
Time
Figure 6.5 Biomass vs time.
Lag phase: In the lag phase there is no increase in microbial density with
time. The length of the phase depends upon changes in the nutrient
composition, age and the size of the inoculum. For instance, if the size of the
inoculum is small, there will be outward diffusion of the nutrients into the
bulk medium. There could be a sudden shift from the old to the new
environment known as adaptability.
Acceleration with exponential phase: This phase is also called the
logarithmic phase. In this phase, biomass increases exponentially with time.
Mathematical expression: We know that the growth rate is proportional to
existing population. Here existing population means the microbial density X,
which has been defined as mass of cells per unit culture volume.
We can write it as
D8
8
DT
D8
or N8 (6.13)
DT
The above equation is called Malthus law.
180 Biochemical Engineering: Principles and Concepts
Rearranging, we get
D8
NDT (6.14)
8
Integrating Eq. (6.14) using the limits X = X0 at t = 0 and X = X at
t = t, and simplifying, we get the resultant as
X = X0eµ(t tlag) (6.15)
Note: t = 0 = tlag
Equation (6.15) shows that the growth is exponential.
Doubling time: At t = td, the time for doubling of biomass, X = 2X0. Using
the expression of the form ln (X/X0) = µt and replacing the values of X and
t by 2X0 and td respectively, we get
LN
TD (6.16)
N
It is an expression for the doubling time of the biomass. In a CSTR, it
is used to characterize the population during the growth.
Stationary phase: The stationary phase starts at the end of the exponential
phase. In this phase, the net growth rate is zero as there is no cell division
where we can say the growth rate is balanced by the death rate. Though the
net growth rate is zero, the cells are still metabolically active and produce
secondary metabolites. During the stationary phase the process called
endogenous metabolism takes place, i.e. cell catabolizes the cellular reserves
for new building blocks and for energy producing monomers.
The cell must expend some energy to maintain an energized membrane
and also for the transport of nutrients for essential metabolic activities. The
metabolic activities refer to the motility and repair of the damaged cells.
This energy expenditure is called maintenance energy.
Conversion of cell mass to maintenance energy or the loss of cell mass
due to the lysis during the stationary phase is given by
D8
8
DT
D8
or +D 8
DT
D8
or +DDT
8
Further integrating, using the limits as X = Xs0 at t = 0 and X = X at
t = t, we get
X = Xs0eKdt (6.17)
where Kd = first-order rate constant for the endogenous metabolism
Xs0 = cell mass concentration at the start of the stationary phase
Biomass Production in Cell Cultures 181
D.
or + D DT (6.18)
.
Integrating the above expression using the limits as N = Ns at t = 0 and
N = N at t = t, we get
N = Ns .eK ¢dt
where K¢d = first-order death rate constant (6.19)
Multiple lag phases: Multiple lag phases may sometimes be observed when
the medium contains multiple carbon sources. Multiple carbon sources can be
in the form of glucose, xylose, sucrose, etc.
During the growth of the cell or a microbe feeding on one particular
carbon source that nears the exhaustion, the cell must divert its energy from
growth to retool for the new source of carbon supply, i.e. second carbon
source. This phenomenon known as diauxic growth which is caused by a shift
in the metabolic patterns in the midst of growth. Figure 6.6 depicts multiple
lag phases during the growth.
Bacterial
density
80
60
40
20
0 2 4 6 8
time (hrs)
glucose xylose
utilization utilization
Figure 6.6 Multiple lag phase.
182 Biochemical Engineering: Principles and Concepts
Growth
rate Tryptophan
Glucose
Glucose concentration/Tryptophan
Complex medium
Glucose (5 g) + Common ingredients
Yeast extract (5 g)
Note: Culture medium can be synthetic or complex based on the
make up.
If glucose is not sterilized separately in an autoclave, then it partially
decomposes to the substances which are toxic. The exact chemical make-up
of the yeast extract is unknown. The others that can be used in place of it
are beef broth, blood infusion broth, corn steep liquor and sewage.
Formulation or make-up is required to support the good growth and the high
yield rates of product synthesis.
1000
500
0 10 20 30 40 50
Temperature
Figure 6.8 Generation time vs temperature.
pH: Protein configurations and the activity of cells are pH dependent. Also
cellular processes, reactions and growth rates depend on pH. For example:
Yeasts grow to the maximum at the pH of 4.05.0. Moulds grow to the
maximum at the pH of 5.07.0. In the case of bacteria, the maximum growth
is seen at the pH of 6.57.0. Therefore, it is seen that different microbes grow
to the maximum at different pH values.
Thermodynamic activity of water and hydrostatic pressure: The thermodynamic
activity of water and hydrostatic pressure influence solute activity in a medium
which in turn affect the dissolved oxygen (DO) concentration.
An increase in DO content in a medium for aerobic processes favours
growth rates.
Cells
DX
Substrates
D S1 Products
System: Fixed
amount of D P1
D S2
cell
material
...
D Sn
D Pm
fixed amount of cell material. Out of the cell are shown the cells produced
(DX) and the products liberated (DP) simultaneously.
It follows the basic equation, i.e.
SS + X ® SP + nX (more cells)
Cell material serves as a catalyst.
Substrates react to produce more cells and metabolic products (typically
an organic compound).
DP
QP
8 DT
qp = Yp/X × µg (6.20)
where X = cell mass concentration (g/L)
mg= gross specific growth rate (hr1)
'0
Yp/X = yield coefficient of product on cell mass.
'8
Figure 6.10 shows a graphical representation of cell mass concentration
or product concentration against time in the case of growth associated
products.
186 Biochemical Engineering: Principles and Concepts
X or P X
P
Time
Figure 6.10 Time vs cell mass concentration/product concentration.
X or P X
Time
Figure 6.11 Time vs cell mass concentration/product concentration.
X or P
X
Time
Figure 6.12 Time vs cell mass concentration/product concentration.
Xi Ci
F F
S
Xif Xi
Sf Ci S
Cif
At the feed side: Let F be the flow rate of the feed. Cif be the
concentration in the feed. Sf be the substrate concentration in the feed
system. In the reactor, the contents are well mixed and change to Xi and Ci
with S as the concentration of substrates.
At the effluent side: Let F be the flow rate, (constant) with Xi, Ci and
S, the same as in the reaction vessel, Monods equation is given as:
Nmax S
N (6.27)
Ks S
Biomass Production in Cell Cultures 189
F 1
(S f S )
VR YX/S N X
F 1
(S f S ) 0 (6.29)
VR YX/S N X
1
D(S f S ) 0 (6.30)
YX/S N X
1
D(S f S ) 0
È Nmax S Ø
YX / S ÉÊ K S ÙÚ X
s
N SX
\ D ( S f S ) max 0 (6.31)
YX/S K s S
F È F Ø
VR
Xf ÉÊ V N ÙÚ X
R
F È FØ
X f ÉN Ù X 0
VR Ê VR Ú
ËN S Û
D . X f Ì max D Ü X 0 (6.33)
Í Ks S Ý
190 Biochemical Engineering: Principles and Concepts
The above equation is called cell mass balance equation. Equations (6.31)
and (6.33) are known as Monods chemostat models.
Biomass
tlag
0 tlag Time
Figure 6.14 Biomass concentration vs time.
Biomass Production in Cell Cultures 191
time. The tangents to the curve are drawn and the point of intersection will
produce the lag time tlag, and shows that virtually there is no increase in the
microbial density or the biomass concentration with time. Lag phase should
always be shorter for a good design.
Initial concentration.
Figure 6.15 Maximum population vs initial concentration.
Logistic curve
X0
Time
Figure 6.16 Microbial density vs time.
D8
In this case, the relation = mX called the Malthus law is modified
DT
as:
dX 2
KX K H X 2 (assuming that inhibition is proportional to X )
dt
dX
KX (1 H X ); X (0) X0
dt
The above equation is called the Ricatti equation.
Here is an inclusion of g on account of inhibition at high biomass
concentration. Inhibition is proportional to the square of biomass
concentration.
On integration, we get
X 0 . e Kt
X
1 H X 0 (1 e Kt )
This is called Logistic equation and the profile shown in Figure 6.16 is called
the Logistic curve.
D;3= D;0 =
R (6.36)
DT DT
It has been discussed in section 3.3.4, the integral MME for batch
reaction kinetics, incorporating material balance and use of MME, we would
finally have
3 3 V MAX T
¹
+M (according to 3.19)
È3 Ø È 3 Ø
LN É Ù LN É Ù
Ê3 Ú Ê3 Ú
Or also can be written as,
È3 Ø
Ê3 Ú
+ M ¹ LN É Ù 3 3 VMAX T
¹
(6.37)
But in a batch process, the flow in and out is both zero. Therefore,
Eq. (6.38) reduces to
{Rate of formation} = Accumulation.
Mathematically, we can write as
D8
RF ¹6 ¹6 (6.39)
DT
or
for biomass,
D8 (6.40)
RF ¹6 N ¹6 ¹ 8 ¹6
DT
In (6.40) as usual, m is the specific growth rate, V is the volume of the
reactor and X is the concentration of biomass prevailing.
If S is considered to be a concentration of limiting substrate, then an
equivalent expression for substrate is:
D;3=
RS ¹6 6 (6.41)
DT
Where rs is the rate of conversion of substrate per unit volume of the
'8
reactor. Using the yield coefficient YX/S = as defined in section 6.9.2.
'3
We can write in terms of Y as
D3 D8
9 (6.42)
DT DT
Y.rs = m.X (as per Malthus law, section 6.6.1)
D3
9¹ N ¹8
DT
D;3=
N ¹8 (6.43)
9 DT
The yield coefficient can also be expressed in its integral form as
'8 8 8
9 983 (6.44)
'3 3 3
On rearranging, we get
S = S0
8 8 (6.45)
983
Let us consider that the growth pattern follows Monods kinetic model,
Biomass Production in Cell Cultures 195
NMAX ;3=
then we have m = . Using (6.40) and substituting for m as shown
+S ;3=
below.
D8
N ¹6 ¹ 8 ¹6
DT
Substitute for m
NMAX ;3= D8
6 ¹8 ¹6
+S ;3= DT
or
NMAX ;3= D8
8
+S ;3= DT
D8 NMAX ;3=
or 8 (6.46)
DT +S ;3=
The condition of fermentation of culture after any time t, is understood
by integrating (6.46) using boundary conditions as, at t = 0, X = X0 and
t = t, X = X.
8 T
+S ;3= D8
Ô
8
NMAX ;3= 8 Ô DT (6.47)
+S ¹9 3 9 8 È8 Ø +S ¹9 È 93 Ø
¹ LN É Ù ¹ LN É Ù T (6.48)
NMAX 93 8 Ê 8 Ú N MAX 93 8 Ê93 8 8 Ú
+S ¹9 3 9 8 È 9 3 3 Ø + S ¹9 È3 Ø
¹ LN É Ù ¹ LN É Ù T (6.49)
NMAX 93 8 Ê 8 Ú NMAX 93 8 Ê 3 Ú
Equations (6.48) and (6.49) can be used to estimate the time needed
during batch growth of MOs.
The main disadvantage is that the changes in biomass and substrate
concentration during batch fermentation are not clearly understood as they
are not explicit in X or S.
Therefore, a trial and error procedure has to be used to find the values
at particular value of time t.
+PRWV 1WVRWV
( (
: :
5 5
#IKVCVQT+ORGNNGT
Let us carry out the material balance for a CSTF. Consider a volumetric
flow rate of feed as F, influent substrate as S0, biomass concentration as X0
(= 0) during initial stages, altogether in a volume V of a fermenter called
the working volume.
Due to reaction taking place, conversion is seen and the conditions
change to S, the substrate concentration, i.e. (S0 ® S) X, the biomass
concentration, i.e. (X0 ® X) while the quantity F remains unchanged.
The overall material balance can be written as
{Material flow in} + {Rate of formation by biochemical reaction}
{Material flow out} = Accumulation.
Biomass Production in Cell Cultures 197
Here we can develop the mass balance for both substrate and biomass.
One should note that substrate is consumed and biomass is formed during the
reaction.
For biomass, referring to the figure,
D8
&8 F RFX6 &8 ¹6 (6.50)
DT
Where rfx is the rate of formation of biomass per unit volume of
fermenter.
For substrate, referring to the figure,
D3
&3 RS ¹6 & ¹ 3 ¹6 (6.51)
DT
(Here rs is ve, as the substrate is consumed).
In the section 6.3.2 we have defined quantity called dilution rate,
&
D= . Here, writing VR = V the working volume or reactor volume and
62
developing further Eq. (6.50) as
D8
&8 F RFX62 &8 ¹6
DT 2
Divide throughout by VR
& 6 & D8 62
8 F RFX 2 8 ¹
62 62 62 DT 62
D8
D Xf + rfx DX =
DT
D (Xf X) = rfx
D8
And further at steady state we can write = 0.
DT
But rfx = m.X (Malthus law)
D(Xf X) = m .X (6.52)
N ¹8
D =
8F 8
8
D = m . (6.53)
8F 8
NMAX ;3=
m=
+S ;3=
198 Biochemical Engineering: Principles and Concepts
We get,
NMAX ;3=
D = (6.54)
+S ;3=
Rearranging the above equation we get,
$+S 3
3 and at steady state, the substrate concentration is
NMAX
$+S
3 (6.55)
NMAX $
Equation (6.55) shows that steady state substrate balance is independent
of feed substrate concentration Sf whereas biomass concentration depends on
the value of Sf.
Further, the yield coefficient
'8
Y = YX/S =
'3
8F 8
YX/S =
3F 3
Rearranging as
Xf X = YX/S (Sf S)
or
X = Xf + YX/S (Sf S) (6.56)
Substitute for S from (6.55) in (6.56)
È ÎÑ $+ ÞÑØ
8 8 F 983 É 3 F Ï S
ßÙ (6.57)
ÉÊ ÑÐ NMAX $ ÑàÙÚ
For a sterile feed Xf = 0,
ÑÎ È $ ¹ +S Ø ÑÞ
X = YX/S Ï3F É ß (6.58)
ÑÐ Ê NMAX $ ÙÚ Ñà
Washout and critical dilution rate Dcritical: From Eqs. (6.55) and (6.58), we
find that substrate and biomass concentration vary inversely with each other.
At low dilution rates, such that D << Ks, then S is small and X is large.
At high dilution rates, it is seen that substrate concentration at steady
state will increase and biomass concentration decreases.
As D nears the value of the mmax, (D » mmax), X becomes infinitesimally
small. This condition is called washout and the value of the D will be critical
called, Dcritical.
The behaviour is depicted in Figure 6.18.
&:
IRGT
5CPF: NKVTGJT
IRGTNKVTG
&
RGTJT
Figure 6.18 Performance curves for CSTF at steady state.
(QT
FKHHGTGPV
-UXCNWGU
&:
&
ÎÑ È $ ¹ + 3 Ø ÞÑ
X = YX/S Ï3F É ß
ÑÐ Ê NMAX $ ÙÚ Ñà
Multiplying the above equation by D
ÎÑ È $ ¹ + 3 Ø ÞÑ
D . X = D.YX/S Ï3F É Ùß (6.60)
ÑÐ Ê N MAX $ Ú Ñà
The above is an expression for cell productivity.
Now when DX is maximum,
D Ë ÎÑ È $ ¹ +S Ø ÞÑ Û
Ì$ ¹9XS Ï3F É ßÜ = 0
D$ ÍÌ ÐÑ Ê NMAX $ ÙÚ àÑ ÝÜ
On differentiating, we get
Solution
Data: Operating volume of a continuous fermenter is 20 litres. Feed is
sterile, Xf = 0; limiting substrate concentration, Sf = 1000 mg/litre, feed rate
F is 5 litre/hr; Ks = 10 mg/litre and mmax = 0.4 and YX/S or Y = 0.45.
To calculate: steady state substrate concentration and steady state
biomass concentration. First calculate the dilution rate for the fermenter i.e.
D = F/VR or F/V
1
D = 5.0/20 = 0.25 hr .
Using the equation for the steady state substrate concentration, i.e.
$+S
3 Eq. (6.55), we get after substitution:
NMAX $
Biomass Production in Cell Cultures 201
Solution
&8
Cell productivity is given as = specific rate of production.
62
Substituting the values in the above equation, we get
5 (442.5)/ 20 = 110.62 mg/litre-hr.
Ë +S Û
Optimum dilution rate is Dopt = mmax Ì Ü [Eq.] (6.61)
Ì +S 3 F Ü
Í Ý
Substituting the corresponding values in the above equation we get,
Dopt = 0.360 hr1.
Solution (Hint)
Data: Substrate concentration given to a culture is 1.0 g/litre; Dcritical is
0.2857 hr1; Change in the dilution rate to 0.0983 hr1 same organism used
but feed concentration is found to be 3.0 g/litre.
To calculate: The substrate concentration in the effluent stream when the
fermenter is operating at its maximum productivity.
Using
N ;3=
D = MAX [Eq. (6.54)]
+S ;3=
Rewriting as
$ 3
NMAX +S 3
$+S
3
NMAX $
Using the above equations for the initial conditions and for the increased
conditions solve for Dopt and S respectively.
C
(TGUJHGGF
'HHNWGPV
%GPVTKHWIG
D
(TGUJHGGF 'HHNWGPV
+PVGTPCNHKNVGT
KPUVCNNGF
E
(TGUJHGGF
'HHNWGPV
'ZVGTPCNETQUU
HNQYHKNVGT
(TGUJHGGF(:H 5H
5GVVNGT
VJKEMGPGT
(
( 4
:5
:
5 %NCTKHKGF
GHHNWGPV( (Y
#GTCVKQP :G5
XGUUGN
4GE[ENGUVTGCO
Substrate balance
D3
FSf + FRS + rSVR (F + FR)S = V . (6.62)
DT
For biomass
D8
FXf + FRXR + rxVR (F + FR)X = V . (6.63)
DT
We know that rx, the rate of formation of biomass is given as rx = mX
(as per Malthus law).
D8
and assuming steady state condition gives = 0
DT
and further saying that feed is sterile, Xf = 0. In the above Eq. (6.63)
F(0) + FRXR + mXVR (F + FR)X = 0
FRXR + mX VR (F + FR)X = 0 (6.64)
Biomass Production in Cell Cultures 205
or
Ks . D [1 R (x 1)] = mmax [S] S . D [1 R (x 1)]
Ks . D [1 R (x 1)] = {mmax D (1 R (x 1)}S
Finally, we have the following equation for substrate concentration:
+S ¹ $; 2Y =
S = (6.70)
NMAX $; 2Y =
To find the biomass concentration at steady state: Using Eq. (6.62)
D3
FSf + FRS + rSVR (F + FR)S = VR
DT
D3
Dividing throughout by VR and putting = 0 (since steady state),
DT
&
D . Sf + rS S =0
62
D . Sf + rS D . S = 0
D[Sf S] = rS (6.71)
'8
Introducing Monods Kinetics with constant yield coefficient, YX/S =
'3
into Eq. (6.71), we have
NMAX3 ¹ 8
D [Sf S] = (6.72)
938 +S 3
On rearrangement, at steady state the biomass concentration is as given
below.
$ ¹983 3 F 3 +S 3
X = (6.73)
NMAX3
NMAX ¹ 3
D = (6.74)
[ 2Y ]+S 3
Biomass Production in Cell Cultures 207
$CRITICAL 3F
(6.75)
NMAX [ 2Y ]+S 3 F
will be arrived at. The above equation confirms that the dilution rate for
wash out will be greater than that for simple CSTF.
(TGUJHGGF(: 5
H H
(: (5
5 :
:
5 :
#GTCVKQP 5
XGUUGN
Note: In Figure 6.22, the first vessel will behave as a simple chemostat
and the performance equation of the same will be valid, while the second
vessel will not receive a sterile feed.
As per Figure 6.22, the feed to the second fermenter will have biomass
concentration X1 and substrate concentration S1, while the first tank (vessel)
will receive a feed with Xf = 0.
+S ¹ $ ; =
S =
^NMAX $; =`
208 Biochemical Engineering: Principles and Concepts
We can write as
+S ¹ $
S1 = (6.76)
[NMAX $ ]
'8
Using yield coefficient YX/S = , we can write for this case as shown
'3
below.
8 8 F
YX/S = (6.77)
3 F 3
But according to Eq. (6.76), we have
+S $
S1 =
[NMAX $ ]
Substitute for S1 in (6.77), we get
8 8 F
YX/S =
È +S ¹ $ Ø
3F É Ù
[N
Ê MAX $ ]
Ú
ÎÑ Ë +S ¹ $ Û ÞÑ
(X1 Xf) = YX/S Ï3 F Ì Üß
Ì N MAX $ ÜÝ àÑ
ÐÑ Í
Since Xf = 0, (during initial stages)
ÎÑ Ë +S ¹ $ Û ÞÑ
X1 = YX/S Ï3 F Ì Üß (6.78)
ÑÐ ÌÍ NMAX $ ÜÝ Ñà
where D1 is the dilution rate for the first vessel.
Carrying out mass balance for second tank, we get
{Flow of material in} + {Rate of formation of biomass}
{Flow of material out} = Accumulation.
D8 .
FX1 + r2.V2 FX2 = V2 (6.79)
DT
D8
FX1 + mX2.V2 FX2 = .V2 (6.80)
DT
At steady state,
D8
= 0
DT
FX1 + m2X2 . V2 FX2 = 0 (6.81)
F(X1 X2) + m2X2 . V2 =0
Biomass Production in Cell Cultures 209
ÎÑ NMAX3
D2 = Ï 8 ÞÑß (6.83)
ÑÐ +S 3 8 8 Ñà
To find the biomass concentration in the second vessel, material balance
over both the fermenters is
X2 = Y [Sf S2] (6.84)
Substituting for X2 from Eq. (6.84) in Eq. (6.83), we get
Î N 3 9 ;3 F 3 =ÞÑ
D2 = ÑÏ MAX ß
+ 3 8 8 Ñ
ÐÑ S à
D2
NMAX3 3 F 3
(6.85)
=
+S 3 È $ +S Ø
É
3 Ù
Ê NMAX $ Ú
Solving Eq. (6.85) for S2 results in
ÎÑ $ $ +S ÞÑ $$+S
(mmax D2) 3 Ï $+S NMAX3 F ß 3 (6.86)
N $ NMAX $
ÐÑ MAX àÑ
Solution (Hint)
Data: Two CSTFs in series arrangement. Operational volume of the first
fermenter is 75 liters and second fermenter is 25 litres.
Sterile feed is given to the first fermenter i.e. Xf = 0; Sf = 3.5 g/litres;
F = 15 litres/hr.
Using the flow diagram (Figure 6.22) we first find out the dilution rates
in both the fermenters.
Dilution rate for the first fermenter, D1 = F/VR = 15/75 = 0.2 hr1.
Dilution rate for the second fermenter, D2 = F/VR = 15/25 = 0.6 hr1.
Now using the Eq. 6.86 and substituting the values and from which we
can find out the value of S2.
ÎÑ $ $ +S ÞÑ $$+S
(mmax D2) 3 Ï $+S NMAX3 F ß 3
N $ NMAX $
ÐÑ MAX àÑ
Substituting the values in the above equation, we get a quadratic form
of equation by which S2 can be determined.
Alternatively adopting Monods equation and using D1 = m1 with usual
notations, for the first fermenter as it behaves like a simple chemostat.
Writing as m1 = D1 = mmax S1/(Ks + S1) or S1 = D1 Ks/(mmax D1), by
which we can find out the steady state concentration S1 of substrate in the
first fermenter.
But first find out the dilution rate, D1 = 0.2 hr1 then finding out S1
using the above equation.
S1 = 0.2 (0.11)/(0.22 0.2) = 1.1 g/litre.
8 8 F
Now using YX/S = (Eq. 6.77)
3 F 3
Taking the yield coefficient as Y = YX/S = 0.4 and Xf = 0 (sterile feed)
we can find out the value for X1 the biomass concentration at steady state.
X1 = Y(Sf S1)
X1 = 0.4 (3.5 1.1) = 0.96 g/litre. (For the first fermenter)
Similarly, mass balance over the second fermenter gives:
D2 = m2X2/(X2 X1) (Eq. 6.82) with usual notations.
The yield coefficient Y = (X2 X1)/(S1 S2), where S2 is the steady state
substrate concentration in the second fermenter.
Rearranging in terms of X2 = X1 + Y (S1 S2), we get:
X2 = 0.96 + 0.4 (1.1 S2)
= 1.4 + 0.4 S2
Biomass Production in Cell Cultures 211
To find Dcritical:
$CRITICAL 3F
=
NMAX +S 3 F
NMAX3
i.e. Dcritical =
F
+S 3 F
Dcritical = 0.22(3.5)/0.11 + 3.5 = 0.213 hr1.
0
$8I
8I (6.88)
$ NI
Similarly, for i = N, the biomass concentration is
$8 .
8. (6.89)
$ N.
$8 .
8 . (6.90)
$ N.
$ ¹ $8 . $ ¹ 8 .
8. (6.91)
$ N. $ N. $ N. $ N.
$CRITICAL 3F
(6.93)
N +S 3 F
0 5
9CUVGUNWFIG
$ . 8
XN = . (6.97)
Ç [$;2 = NI ]
I
&2
where R = recycle ratio = (recycle stream)
&
We know that the biomass concentration entering the first tank is not
zero. Then,
&2
8 F 82 (6.98)
&2 &
&
, since R = 2
2
8 F 82
2 &
2
$ . 82
2
Therefore XN = . (6.99)
Ç
I
[$;2 = NI ]
. 2
$CRITICAL 82
2
XN = . (6.100)
Ç [$CRITICAL ;2 = NCRITICAL ].
I
82
or using x = and rearranging to give D, we get
8
NCRITICAL
$CRITICAL .Û (6.101)
Ë ÎÑ Y2 ÞÑ
2 Ì Ï ß Ü
Ì ÑÐ 2 Ñà Ü
ÍÌ ÝÜ
NMAX ¹ 3
where NCRITICAL
F
+S 3 F
Substituting for mcritical in Eq. (6.101), we get
Biomass Production in Cell Cultures 215
NMAX3 F
+S 3 F
$CRITICAL . Û
Ë (6.102)
ÎÑ Y2 ÞÑ
2 Ì Ï ß Ü
Ì Ü
ÌÍ ÑÐ 2 Ñà ÝÜ
F8
(TGUJHGGF
( 'HHNWGPV
: H (G
5 H 5
F5:
F: :G
5 5G
:
(+
:+
5+
<
+PQEWNWO
F<
Figure 6.25 Plug flow fermenter.
D8 .
(F0 + FI)X + rf .dV (F0 + FI) (X + dX) = V (6.103)
DT
If Ac is the cross-sectional area of fermenter, then we can write
dV = Ac dZ
D8
and further at steady state writing ¹6 , we get
DT
(F0 + FI)X + rf . Ac dZ (F0 + FI)(X + dX) = 0
Simplifying further
F0 . X + FI . X + rf . Ac . dZ (F0 . X + F0 . dX + FI .X + FI . dX) = 0
rf . Ac . dZ F0 . dX FI . dX = 0
rf . Ac . dZ FA . dX = 0 (6.104)
Since after mixing the inoculum with feed, let us say an entry stream of
flow rate FA, with XA and SA is produced.
Rearranging and later integrating
XE Z
D8 !C
Ô
X!
RF &! Ô D:
(6.105)
XE
D8 !C
Ô
X!
RF &!
¹: H
(6.106)
3
Ô RS
H (6.108)
!
Biomass Production in Cell Cultures 217
where
&3 F &) 3 )
3! (6.109)
& &)
Considering a constant yield coefficient Y, the final equation is
+S9 3!9 8 ! Ë 9 3! 3E Û +S ¹9 Ë3 Û
¹ LN Ì Ü ¹ LN Ì E Ü H (6.110)
NMAX 93! 8 ! ÌÍ 8! ÜÝ NMAX 8! 93! ÌÍ 3! ÜÝ
F8
(TGUJHGGF
( 'HHNWGPV
: 5
F5:
F:
H
5
5H :
(G
:G
<
5G
F<
'HHNWGPV
5
4GE[ENGUVTGCO
(4 :454
9CUVGUNWFIG
(Y 5Y5G: Y:4
where
&2
R = = recycle ratio
&
The performance of a plug flow fermenter is
XE Z
D8 !C
Ô
8!
RF Ô & 2 D:
(6.114)
The material balance for substrate about the mixing point of fresh feed
and the recycle stream is
Sf . F0 + SR . FR = SA(F0 + FR) (6.116)
3 23E
where 3! (6.117)
2
Similarly, for biomass, the material balance
Xf . F0 + XR . FR = XA(F0 + FR) (6.118)
82 &2
Using x = ; so that XR = xXe and R = , the recycle ratio; above
8E &
equation can be rearranged as:
8 E Y2
8! (6.119)
2
now material balance over element of dV is
D3 .
(F0 + FR)S + rS . dV (F0 + FR)(S + dS) = V (6.120)
DT
&2 D3
using R = and at steady state, it is written as
& DT
F0(1 + R) dS + rS . dV = 0 (6.121)
then
NMAX ;3=8 !!#
F0(1 + R)dS = ¹ D: (6.124)
9 +3 ;3=
Since S = SI when z = 0 and S = Se at exit, then for reactor of length Z,
:
S
+S 3 D3 NMAX 8 !!#
Ô 3
9& 2 Ô D: (6.125)
S!
yields the following:
È3 Ø NMAX 8 !!#
3 ) 3E +S LN É ) Ù D: (6.126)
Ê 3E Ú 9& 2
Using the yield coefficient
'8 8E 8 )
Y =
'3 3) 3E
NMAX 8 !!# È3 Ø
8E 8 ) D: 9+S ¹ LN É ) Ù (6.127)
& 2 Ê 3E Ú
(TGUJHGGF(: :
5 5
H H
(:5
#GTCVKQPXGUUGNYKVJ
CYQTMKPIXQNWOG8
Figure 6.27 The chemostat (simple configuration) for estimation of kinetic parameters.
NMAX3 8
D(S0 S) = (6.129)
9 +S 3
Writing in the reciprocals form for the above equation, we get a linear
form of expression as
8 +S9 9
(6.130)
$3 3 NMAX3 NMAX
Using Eq. (6.131) to plot X/D (S0 S) versus 1/S it is seen that a
straight line is obtained as shown in Figure 6.28.
:&
5 5
5
Figure 6.28 Graph showing X/D (S0 – S) vs. 1/S for a chemostat culture.
In order to find the value of Ks evaluate the slope of the straight line
and calculate the intercept.
To find the value of mmax, at first we should calculate the value for Y.
Ignoring the maintenance requirements of the culture, we find that the yield
coefficient for growth is equal to the overall observed yield coefficient and
is given as
8 8
9 (6.131)
3 3
But since the feed is sterile, we can write X0 = 0
8
Y = (6.132)
3 3
Note: The mean value of Y calculated at each dilution rate used in the
experiments will allow mmax to be determined from the graph.
In a more realistic way it is better we calculate the specific growth rate
considering endogenous respiration Kd as defined in section 6.6.1 and the
equation is given below.
222 Biochemical Engineering: Principles and Concepts
NMAX3
N +D (6.133)
+S 3
When dilution rate is equal to specific growth rate, i.e. D = m, we can
write
NMAX3
$ +D (6.134)
+S 3
Therefore, Eq. (6.129) can be rearranged to give:
NMAX3 3 3
9$ (6.135)
+S 3 8
3 3 +D
(6.136)
8 9$ 9
Using the above equation to plot (S0 S)/X versus 1/D, we find that a
straight line is produced which has a slope of Kd/Y and an intercept 1/Y
(Figure 6.29).
5 5:
&
Figure 6.29 Graph showing (S0 – S)/X vs. 1/D for a chemostat culture.
Solution: Hint: First, using the information given in the table above
calculate the values for (S0 S)/X; 1/D; 1/S; X/D (S0 S) using S0 = 500
mg/litre, the fresh fed concentration.
Then represent in the form of a table respectively as indicated.
Next using the values (S0 S)/X versus 1/D, plot the graph as shown
below in Figure 6.30.
5 5:
5NQRGQHVJGNKPG
+PVGTEGRV
&
Figure 6.30 Example 6.1: (S0 – S)/X vs. 1/D.
From the graph find the intercept and slope of the line.
From the intercept, using intercept = 1/Y we can find out the value for
Y, the yield coefficient.
224 Biochemical Engineering: Principles and Concepts
Using the slope of the line = Kd/Y, find out the value for Kd, the
endogenous respiration coefficient (per hour).
Next plot the graph of X/D (S0 S) versus 1/S as shown in Figure 6.31.
5NQRGQHVJGNKPG
+PVGTEGRV
5
Figure 6.31 X/D (S0 – S) vs. 1/S.
From the above figure, find the intercept = Y/m, in terms of hours.
Therefore maximum specific growth rate mmax (per hour) = Y/intercept.
+S9
Using the slope = (mg per litre per hour)
NMAX
Find the value for Ks, the Monods constant in terms of mg/litre.
The clear explanation of growth kinetics of a cell culture will involve the
identification and recognition of the structured nature of each cell and the
segregation of cultures into the individual cells or units, (refer section 6.2 for
structured, segregated and unstructured and unsegregated models) that may
differ from each other.
We have discussed in the preceding sections about the models being
structured, segregated and unstructured and unsegregated in describing the
cell population kinetics. Also we know that the ones which are structured and
segregated pertain to the actual case and are more realistic, while
unstructured and unsegregated are considered to be the ideal ones.
Structured and segregated models are complex for computations,
although being realistic in nature. The degree of realism and complexity
needed in a model depends upon what is being described.
An unstructured model assumes fixed cell composition which is as good
as assuming the state of balanced growth. The concept of balanced growth
as understood is valid primarily in a single stage and steady state conditions
of a continuous culture and also in the exponential phase of growth of batch
cultures. However, it fails to validate during the transient conditions.
Overall, the use of either the structured or unstructured and segregated
or unsegregated depends upon the conditions or the situations prevailing, but
all have some significance.
In the succeeding paragraphs, we find the use of certain models to predict
the specific growth rate of cultures.
)TQYVJ
TCVGRGT
JQWT
5CVWTCVKQP
5WDUVTCVGEQPEGPVTCVKQP5OINKVTG
Figure 6.32 Saturation kinetics profile for culture growth.
226 Biochemical Engineering: Principles and Concepts
NMAX3
NG
È 3 Ø
+S É Ù 3
Ê +) Ú
NMAX3
NG
È 0 Ø
+S É Ù 3
Ê + 0) Ú
Inhibition by toxic compounds: The toxic compounds are likely to enter the
reaction vessel or are generated during the release of metabolites and finally
inhibiting the growth rate of the cultures and microbes.
The expressions suggested below are all analogous to the enzymatic
inhibition mechanisms.
228 Biochemical Engineering: Principles and Concepts
NMAX3
NG
È Ø
É + ÙÈ ) Ø
S
É 3 Ù É Ù
É ) Ù Ê +) Ú
ÉÊ +) ÙÚ
NMAX3
NNET +D
+S 3
Note: The logistic model or approach that refers to the inhibition due to
high biomass concentrations is discussed in section 6.9.8.
The growth models for such cases should include simultaneous diffusion
and nutrient consumption within the microbial pellet at large pellet sizes.
Interestingly, it is also observed that these filamentous organisms also
grow on the moist solid surfaces, so called attached growth. Indeed such
growth is a complicated process that involves not only the growth kinetics
but also the diffusion of nutrients and toxic metabolites.
To elaborate the description of filamentous organisms we consider that
a microbial pellet is formed in a submerged culture of a mould colony
growing on the surface of an agar. This is showing growth that varies linearly
with time.
A microbial pellet of radius r is growing as a function of time t. It is to
be assumed here that there are no mass transfer limitations during the
process.
We can write the above statement as:
DR
KPELLET CONSTANT
DT
The growth rate of a mould colony can be further expressed as:
D- DR
S Q R
DT DT
D-
S Q R KPELLET
DT
or
D-
V-
DT
where v = kpellet (36 pr)1/3
Integrating the above equation, we get
- È - VT Ø
Ê Ú
where M0 is the initial biomass.
This is congruent to (vt/3)3.
The initial biomass M0 is very small compared to M and therefore M
varies with cube of time factor, i.e. t3.
Note: A detailed discussion of the above two models is beyond the scope
of this book.
Introduction to fed batch reactors: In batch type reactors, it is seen that the
input of the raw materials is given and the products formed are removed,
forming a batch. It is always felt necessary that the liquid streams be added
to a batch fermenter as the reaction is progressing.
In compliance with this, the nutrients are added continuously or semi-
continuously, while the effluent is removed discontinuously. This is also called
a repeated fed batch culture.
Addition or feeding during the process aids in precursors for the desired
products i.e. the desired product(s) can be obtained as these are carried
forward for withdrawal.
In addition to this, regulating compounds as inducers and intermittent
substrates are also fed at a desired point in the batch operation so as to
maintain low nutrient levels to minimize catabolite repression and substrate
inhibition.
If the substrate is inhibitory in nature, intermittent addition of substrate
improves the productivity of the process by keeping the substrate
concentration very low. When a liquid feed stream is added and enters the
reactor, the culture volume also gets altered.
Therefore, fed batch reactors are also referred to as the semicontinuous
or variable volume continuous culture reactors.
The process of fed batch culture is shown in Figure 6.33 schematically.
Biomass Production in Cell Cultures 231
(5
5VCTVQHVJG
DCVEJRTQEGUU
#GTCVKQPXGUUGN
8 :52
(5
(KNNKPIQT
CFFKPIUVCIG
#GTCVKQPXGUUGN
8 :52
*CTXGUVKPI
QHEGNNUUVCIG
#GTCVKQPXGUUGN
8:52
D
(VR) = F(t) (6.139)
DT
Differentiating Eq. (6.137) and then substitute for dVR/dt using (6.139),
we get on final rearrangement as
D# I & T
# IF # I RFI (6.140)
DT 62
The above equations are used to assess the fed batch reactor
performance.
SUMMARY
In this chapter, we learnt the following:
· Cell growth kinetics can be modelled. They can be structured or
unstructured. Unstructured models quantify cell mass as a single
component and cannot describe the transient behaviour.
· In batch cultivation, a population of cells typically exhibits several
different growth phases.
· Lag phase, exponential phase, stationary phase and death phase are
four growth phases.
· Products formed by cells can be related to the batch-culture growth
cycles. Primary products are growth associated, secondary products
are non-growth associated and are made in the stationary phase.
· Some products are both growth and non-growth associated
components.
Biomass Production in Cell Cultures 233
EXERCISES
6.1 Define the following terms:
(a) Biomass
(b) Cell culture
(c) Synthetic and complex mediums
(d) Cell population
6.2 Explain the important parameters, phenomena and interactions
between the growth medium and the cell population which determine
cell population kinetics.
6.3 Elucidate different perspectives for cell population kinetic
representations.
6.4 In an ideal batch reactor, for kinetic measurements, show that the
rate of formation of a component is the change in its concentration
with time.
6.5 Define dilution rate in a CFSTR and show that D = F/VR.
6.6 Describe Monods growth kinetics and define specific growth rate.
6.7 Describe the formulation of synthetic and complex mediums for cell
cultures.
234 Biochemical Engineering: Principles and Concepts
6.8 What are the environmental factors that affect growth kinetics? Explain.
6.9 Describe transient growth kinetics. With a neat sketch, explain the
phases of growth.
6.10 Write a note on the following:
(a) Multiple lag phases
(b) Doubling time
(c) Yield and maintenance coefficients
(d) Chemostat
(e) Growth, non-growth and mixed growth associated products
6.11 During the batch growth of microorganisms, derive an expression for
time needed in terms of substrate concentration.
6.12 In the continuous culture systems, explain the following:
i(i) Cell productivity and
(ii) Wash out and critical dilution rate.
6.13 What are the strategies adopted in the recycling of biomass using a
CSTF? Explain.
6.14 Using a settler-thickener arrangement in a CSTF with recycle, arrive
at the following expressions:
i(i) Substrate concentration at the steady state.
(ii) Biomass concentration at the steady state.
Further prove that the dilution rate for wash out in a CSTF with
recycle is greater than that in a simple CSTF with no recycle.
6.15 Derive the expressions for CSTF arrangement in series.
6.16 The steady state substrate and biomass concentrations for a continuous
stirred tank fermenter operated at various dilution rates are shown in
the following data. Given that the fresh feed concentration is 695 mg
per litre, calculate the values for Monods constants, the yield
coefficient and the endogenous respiration coefficient.
Biological Reactors
A handful of basic bioreactor designs are used to produce a wide range of
products, from antibiotics to food products to fuels. Here is how to pick the
best options for our application. Traditionally, microbiologists have played
a dominant role in the development of bioreactions with the assistance from
those in multiple disciplines, including biochemists, geneticists and chemical
engineers. Fermentation process that is in use since antiquity is a precursor
to modern bioreactions. However, major advancements of the last half
century have much to do with the technology as with biology.
In this chapter, emphasis is given on the illustration of the relevance of
established chemical engineering practices and processes as they apply to
todays bioreaction engineering, as chemical engineers make further inroads
into a field once thought to be the sole domain of biology-based scientists.
Further, it discusses key engineering issues in bioreactor design and
operation, focusing on similarities between traditional chemical reactor
engineering and bioreaction engineering and even key differences that must
be taken into consideration for successful bioreactions are also stated.
Various types of reactors used in the biochemical industry with their
significance and utilities are also met with. Alternate reactor configurations
are briefly touched upon with the concept of medium formulation and aseptic
conditions.
236
Biological Reactors 237
Primary products for industrial use are ethanol, citric acid, acetone,
butanol, lysine, vitamins, etc.
Secondary metabolites: Secondary metabolites are produced from
intermediates and are products of primary metabolism. These may be toxic
and possess the antibiotic properties.
Note: Not all microorganisms produce secondary metabolites, only a few,
filamentous microorganisms like fungi, plant cells, etc.
Process 2: During this type of process a cell mass is produced. Bakers
yeast that is used in baking industry, is an example of a produced cell mass.
The others include single-cell proteins for food sources.
Process 3: This type of process modifies a compound that is added to
the fermentation process and is referred to as biotransformation. The process
can be by way of dehydration, oxidation, hydroxylation, amination, etc. By
way of biotransformations, the products produced are steroids, antibiotics,
hormones, etc.
Fermentation vessel
Filtered air
Sparger
Downstream or Cell
recovery section
Seed tank
Fermenter
Steam C
J
F Steam
E G
D A B
I H
Steam trap
With a proper and close check on the steps mentioned above, aseptic
inoculations can be achieved for long-scale fermenters.
G M M G G
With overflow Overfilled
G M G M
G G
LR SZ
M
M
G
MMotor SBbaffles MMotor
GGas(Air) LRconduit tube SZFoam breaker LRconduit tube
G G M GGas (Air) LSelf-gas agitator
G
G G
F
M W W
G F
MDrive motor
G M G Pulsation WRoller
G G
Figure 7.3 Alternate bioreactor configurations showing the use of mechanically moved
internals for energy input. (Source: Bailey and Olis, Biochemical Engineering
Fundamentals, 2nd ed., McGraw-Hill, 1986.)
Biological Reactors 243
Exhaust
Gas
Gas
Supply
Trays
Feed
Gas
supply Biomass
recycle
Valve Product
Pump
Separator
Figure 7.5 Stirred tank bioreactor.
As the reaction progresses, the bubbles produced by the air supply are
broken up by the agitator as they travel upwards. Many types of agitators
are used, with the most common being a four-bladed disk turbine. The
turbine is a cylindrical structure with the baffles that serve the purpose as
usual. Concentric to it is an agitator driven by a motor at the top that helps
in vigorous mixing. At the bottom is given gas supply so as to aerate reactor
contents. After the reaction is complete, the contents are drained from the
bottom through the drain valve and fed to the pump. This is given to the
separator that segregates into the product and the biomass to be recycled.
From the top, exhaust gases emanate.
Stirred tank bioreactors have been modified to reduce shear rates on the
cells in suspension. Sail-type and axial-flow hydrofoil agitators have been
developed and used for cell suspension cultures. The agitation rate in these
reactors is of the order of 10 to 40 rpm, providing a low shear rate.
Exhaust gas
Gas supply
Valve
Product
Figure 7.6 Concentric draught tube reactor.
the tank. The tube can be designed to serve as an internal heat exchanger,
or a heat exchanger can be added to an internal circulation loop.
Airlift systems provide some advantages against more conventional
bioreactors as fermentors:
· Simple design with no moving parts or agitator shaft seals for less
maintenance, less risk of defects and easier sterilization
· Lower shear rate, for greater flexibilitythe system can be used for
growing both plant and animal cells
· Efficient gas phase disengagement
· Large, specific interfacial contact area with low energy input
· Well-controlled flow and efficient mixing
· Well-defined residence time for all phases
· Increased mass transfer due to enhanced oxygen solubility achieved in
large tanks with greater pressure
· Large volume tanks possible, increasing the output
· Greater heat removal against conventional stirred tanks.
The main disadvantages are as follows:
· Higher initial capital investment due to large-scale processes
· Greater air throughput and higher pressures needed, particularly for
large-scale operations
· Low friction with an optimal hydraulic diameter for the riser and the
downcomer
· Lower efficiency of gas compression
· Inherently impossible to maintain consistent levels of substrate,
nutrients in oxygen with organisms circulating through the bioreactor
and conditions changing
· Inefficient gas/liquid separation and foaming occurs
248 Biochemical Engineering: Principles and Concepts
Gas supply
Valve
Product
Figure 7.7 Airlift external loop reactor.
The AELR has some advantages over standard airlift reactor and they
are as follows:
· Effective heat transfer and efficient temperature control
· Low friction with an optimal hydraulic diameter for the riser and the
downcomer
· Well-defined residence time in the individual section of the AELR
· Increased opportunity for measurement and control in the riser and
the downcomer
· Independent control of the gas input-rate and liquid velocity by a
throttling device between the riser and the downcomer
It has induced circulation that directs air or liquid throughout the vessel.
Loop reactors have intermediate characteristics between the bubble columns
and stirred tanks. The motion of gas carries fluid and cells up a draft tube.
At the top, the gas disengages from the liquid, and the degassed liquid
descends in the reactor. Such type of reactors can handle somewhat more
viscous fluids than the bubble columns, and coalescence is not so much of a
problem. The largest agitated fermentor ever built by Imperial Chemical
Industries (ICI) is an airlift design for the production of single-cell protein.
One (or more) reactant is provided in each feed liquid and gas phase, so
that biochemical reaction depends on contacting of liquid, containing a
sparingly soluble reactant from the gas phase, with the catalyst surface.
The performance of such reactors is substantially influenced by the
physical state of gas-liquid flow through the fixed bed and by the associated
mass transfer processes.
The important characteristics of such a reactor are the surface area of
the packing, the efficiency of wetting the catalyst by the flowing liquid phase,
the gas-liquid flow pattern, mass transfer of sparingly soluble reactants from
the gas to liquid phase, mass transfer of both reactants to the catalyst surface
and, in case of a porous or permeable catalyst, diffusion of reactants to the
intra particle catalyst sites.
In Figure 7.8, is shown a schematic diagram of a trickle bed reactor.
Exhaust gas
Feed
Product
Gas Pump
supply
Figure 7.8 Trickle bed reactors.
The trickling biological filter is a trickle bed reactor and is used for
wastewater treatment. Trickle bed reactors are used in the production of
vinegar and also in petroleum and petrochemical industry for hydrocracking,
hydrotreating and other multiphase reaction processes.
The bioreactors will be integral to the development of many high-value
products and the replacement of existing chemical-based commodity
processes. The proper selection and design of a bioreactor will determine an
optimal commercial bioprocess and corresponding capital investment.
4GE[ENGUVTGCO $NGGF
(GGF
/GODTCPG
%GNNUCTGKOOQDKNK\GF
2GTOGCVG
2WOR
(GGF
2TQFWEV
2GEVKPCUGU
Figure 7.10 Membrane as a contactor.
#SWGQWURTQEGUU
$QWPFCT[ UVTGCO
CSRJCUG
5QNWDNGUWDUVTCVG5
%QPEGPVTCVGF
UVTGCO
22TQFWEVKP
CSWGQWUUQNWVKQP
+OOQDKNK\GFQTICPKE
NKSWKFOGODTCPG
'P\[OGCEVKXCVGF
OGODTCPG
Figure 7.11 Membrane as a contactor and separator.
SUMMARY
In this chapter, we learnt the following:
· A bioreactor is a system in which a biological conversion is effected.
· A bioreactor should not be regarded as an isolated unit, but as part
of an integrated unit operation with both upstream (preparation) and
downstream (separations) unit operations.
· There are different types of bioreactors/bioreactingbatch,
continuous semicontinuous, surface/tray submerged, airlift loop and
trickle bed units.
Biological Reactors 253
EXERCISES
7.1 Discuss the features of bioreactors and explain how these are different
from the chemical reactors?
7.2 Explain the products of bioreactions.
7.3 Highlight key issues to be considered in the design and operation of
bioreactors.
7.4 With the help of a neat flow sheet, explain the components of a
typical fermentation process.
7.5 Discuss with the help of a neat sketch the operation of a typical
aseptic aerobic fermentation process.
7.6 Enlist the steps of the preparation of inoculum.
7.7 Write on the alternate bioreactor configurations.
7.8 With a neat sketch, describe the working and maintenance of a tray
type bioreactor.
7.9 Write a note on the following:
(a) Trickle bed reactors
(b) Concentric draught tube reactors
(c) Airlift reactors
(d) Stirred tank bioreactors
7.10 With advantages and disadvantages, explain the different
configurations of membrane bioreactors.
Chapter 8
Fermentation Technology
Traditional Processes and Products
8.1 FERMENTATION
Our knowledge of the use of fermentation is as old as the civilization itself.
Right from the leavening of bread, the rising of wet ground, preparation of
idli and dosa, to the transformation of milk to cheese and the making of wine
and vinegar are all well-understood current practices wherein we have
continually used microorganisms even without being aware of it. Today, this
knowledge is intentionally used to devise and design innovative and practical
applications of microbial activities. In the succeeding discussions, emphasis
is placed on the processes and technologies used in the production of various
fermentable products.
254
Fermentation Technology—Traditional Processes and Products 255
Air
Tricklers
Beech wood
shavings
Air
Pump
Vinegar
Perforated bottom
Figure 8.1 Frings vinegar generator.
Later, the mix is collected at the bottom and can be recirculated over
the shavings that result in further oxidation of alcohol to acetic acid, until
vinegar of desired strength is obtained.
The liquid is then stored anaerobically to prevent further degradation.
Ageing takes place during the storage. Esters are formed and the harsh
flavour disappears. Further, vinegar is clarified by filtration, pasteurized and
bottled.
An adequate supply of air must be available throughout the chamber.
It is also necessary to keep the temperature between 15 and 35°C.
Fermentation Technology—Traditional Processes and Products 257
Lactic acid: Lactic acid was first isolated from sour milk in the year 1780.
It has two optically forms called D- and L-lactic acids. Most of the lactic
acid is produced by fermentation process. For the production of lactic acid,
several carbohydrate substances like corn, starch from potatoes, molasses and
whey are used.
Let us emphasize the production of lactic acid using whey. Large
quantities of whey constitute a waste during the manufacture of certain dairy
products. Whey is a satisfactory medium for the growth of certain bacteria
as it contains growth favouring constituents like carbohydrates (lactose),
nitrogenous substances including vitamins and salts. Lactobacilli are the
microorganisms that are suitable for this process, more specifically,
Lactobacillus bulgaricus.
Figure 8.2 depicts the production of lactic acid from whey.
Filtration, evaporation
and purification
Starter culture in milk Whey with culture Whey in tank
CO2
Water
CaSO4 Filter
H2SO4 Evaporator
Filter
Seeding tank
Production vessel Sludge Decomposer Crude lactic acid
to purification
Figure 8.3 Flow sheet for the production of lactic acid.
The first step is to increase the temperature to about 80100°C and the
pH to 1011. Due to this, the organism is killed and coagulates proteins,
solubilizes calcium lactate and degrades some of residual sugar. The liquid
is then filtered to remove the biomass, and sulphuric acid is added to obtain
lactic acid. Calcium sulphate is removed by filtration and lactic acid is then
concentrated by way of evaporation. Further, crude lactic acid is taken for
purification.
Recovery of lactic acid from the fermentation broth constitutes a
significant part of the production cost.
The major use of lactic acid is in food as an acidulant and preservative.
It is also used as a chemical intermediate to produce other chemicals and in
pharmaceutical industry.
In the production of lactic acid, industrial processes are operated
batchwise. The features of the fermentors and process conditions for the
various organisms employed are discussed as follows:
Fermentors are made of stainless steel and are equipped with heat
transfer coils. Prior to the addition of a pasteurized medium to the vessel,
it is steam sterilized to see that there is no unwanted microbial growth. In
order to prevent the settling of calcium carbonate, slow agitation is provided
with top mounted mechanical stirrers. For each industrial producer, the
fermentation conditions are different:
· T = 4560°C and pH = 56.5 for L. delbruckii
Fermentation Technology—Traditional Processes and Products 259
Ethanol
concentrated
Ethanol vaporizer
Feed
Stillage
stripper
Concentrated stillage
Fermenter
The above process flow sheet depicts a bio-still process for the production
of ethanol. Continuous operations with cell recycle and a distillation column
for ethanol separation are employed. A stainless steel fermenter with
mechanical agitation is operated in the continuous mode, and the effluent is
centrifuged for yeast separation. Yeast is used in the manufacture of ethanol.
Part of the separated yeast is recycled to the fermentor as it has two facets
of importance: the yeast recycle provides high conversion rates and liquid
recycle decreases the amount of wastewater generated and also dilutes the
feed sugar concentration down to non-inhibitory levels.
Yeast immobilization within porous or polymeric matrices will result in
high cell concentration inside the reactor as a result of which loss of yeast
is reduced. This increases the rates of ethanol production. The immobilized
cell reactors are in the form of packed columns or fluidized beds. Certain
flocculating yeast strains that can settle rapidly are also used in the
fermentors in order to obtain high cell concentrations.
Conventional ethanol fermentations operate in the batch mode under the
aseptic conditions. But, the use of continuous mode is preferred as the risk
of contamination by microbes is minimized with the use of continuous
sterilization reactors.
The reaction accomplished by the yeast is as follows:
yeast/enzymes
C6H12O6 2C2H5OH + 2CO2
Fermentable carbohydrate Ethanol Carbon dioxide
At the end, to get the ethanol of very high purity, azeotropic distillation
is employed. As we know that ethanol forms an azeotrope with water at 95.7
wt% or 89 mol% ethanol. The recovery by distillation is done in two steps.
First, a three-column conventional distillation train produces a high alcohol
product stream. For anhydrous alcohol production as needed for motor fuel
usage, this binary solution is mixed with benzene that acts as an entrainer
to break the binary azeotrope and allowing 100% recovery of ethanol.
Figure 8.5 depicts the process of distillation to get ethanol.
Alcoholic beverages and their manufacture represent one of the largest
industrial applications of microbial activity. In brewing both carbon dioxide
and ethanol formed as a result of fermentation become desirable products.
Further, in brewing, the growth of yeast in a sugary medium should take
place under anaerobic conditions or else the process of fermentation gets
inhibited.
Wine and beer are produced in large quantities as alcoholic beverages.
Of course, the raw materials and the processes employed in both are different.
Wine: Wine is made mostly from the juice of grapes and to a lesser
extent from the juices of other fruits. The juices containing a large
concentration of sugar cane, on fermentation by yeast, be converted to
ethanol and carbon dioxide.
Fermentation Technology—Traditional Processes and Products 261
EtOH-H2O
azeotrope recycle
Reflux drum
Feed
Makeup
C6H6
Steam
Ethanol Water
Azeotropic distillation Benzene recovery Water removal
Figure 8.5 Azeotropic distillation of ethanol.
Grapes are crushed to extract juice. The juice itself is a strong solution
as it contains many moulds, yeasts and bacteria from the surface of grapes.
This solution is treated with sulphur dioxide to remove microbial population
and inoculated with starter cultures of the yeast Saccharomyces ellipsoideus.
The duration of fermentation ranges from a few days to about 15 days.
Temperatures in the range of about 1015°C, are considered to be the best
for fermentation.
The product is then cured to allow the wine to age. This is a slow
complex process and a moderate supply of oxygen is required.
Only a properly aged wine will have a desirable aroma and flavour and
such wine alone is bottled. Dry wine has a little or no unfermented sugar.
Sweet wine has unfermented sugar giving it a sweet taste.
Rum: Rum is prepared by fermentation of black strap molasses
containing 1214% sugar with S. cerevisiae, to which ammonium sulphate
and phosphate are added. After fermentation, the alcohol is distilled and
bottled.
Beer: Beer is prepared from grains like barley, rice and maize as
principal carbohydrate, starch is present in them. Yeasts cannot ferment
starch but only sugar. Beer is manufactured by the breaking down of starch
of sprouted barley into suitable sugar by amylase obtained from other sources.
Fermentation is allowed to start thereafter and is allowed to proceed
slowly. The characteristic bitter flavour of beer is produced by the addition
of flower buds of the hop plant.
262 Biochemical Engineering: Principles and Concepts
Distillation tower
Fractionators
Dried stillage
Butanol
Acetone
Drier
Ethanol
Cooler
Fermenter
Beer still
Figure 8.6 Production of acetone/butanol.
drops from 6.5 to 4.5. The second phase exhibits cessation of growth and
organisms convert acetic and butyric acids to neutral acetone and butanol.
At the end of fermentation the pH is around 5.0. In order to improve the
solvent yield in acetone/butanol fermentations, and butanol production
following steps are taken up: addition of acetic and butyric acids and
moderate to high agitation (300 rpm) during the acid phase followed by low
agitation (25 rpm) during the solvent phase respectively. Besides this, the
removal of inhibitory products (butanol/acetone) by adsorption employing
activated carbon is done. This improvises the fermentation productivity.
At the end of solvent phase to recover acetone and butanol, broth is
transferred to a beer still that concentrates the solvents. Further, solvents are
separated by distillation and fractionation and stillage is dried. Finally, three
streams, butanol, acetone and ethanol are recovered.
Biomass 2% w/v
Lime
Brother vessel and Crystallization
H2SO4 stirring
Wash water
H2O
Citric acid
Evaporation Drying of citric acid product
Sludge of CaSO4
Figure 8.7 Production of citric acid.
The precipitate is then washed and treated with dilute sulphuric acid,
yielding an aqueous solution of citric acid calcium sulphate precipitate.
After bleaching and crystallization, either anhydrous or monohydrate
citric acid is obtained. Later, centrifugal recovery is done followed by drying
crystals. Solvent extraction is another option for the recovery of citric acid,
although it is not used commercially.
Bakers yeast: The use of yeast as a leavening agent in the baking process
dates back to the very early history of the Jews. In modern baking practice,
pure cultures of selected strains of S. cerevisiae are mixed with the bread
dough to bring about the desired changes in the texture and the flavour.
The desirable characteristics of strains selected for the commercial
production of bakers yeast include the ability to ferment sugar in dough and
to grow rapidly and should be relatively stable.
Figure 8.8 depicts the steps in the production of bakers yeast.
In the production of bakers yeast, the medium that contains molasses
and cornsteep liquor is inoculated with a stock strain, while the pH of the
medium is brought to acidic value (45). This aids in the retardation of
bacterial growth, the unwanted ones. The total medium is incubated during
which aeration is provided. At the end of incubation period, the yeast cells
are harvested by adopting centrifugation and washing of the cells by
suspending in water and again centrifuging the cells out.
The cells are finally recovered by filtration process using a filter press.
Over the press, cells are present and these are treated with small amounts of
edible oil which serves as a plasticizer in giving the form to the cells as they
are molded into blocks.
Fine chemicals: Fine chemicals include the bioproduction of high value
molecules like antibiotics, vitamins, hormones, enzymes, monoclonal
antibiotics, etc.
Antibiotics: Penicillin was the first antibiotic to be produced industrially.
The major steps in the commercial production of penicillin are as follows:
· Preparation of inoculum
· Preparation and sterilization of the medium
· Inoculation of the medium in the fermenter
· Forced aeration with sterile air during fermentation
· Removal of the mold mycelium after fermentation
· Extraction and purification of penicillin
Antibiotic production yields either a bulk salt form, e.g. sodium
penicillin or a more purified precipitate, procaine penicillin for clinical use.
Figure 8.9 shows the method to manufacture and purify crude penicillin, to
get procaine penicillin.
Penicillin is a secondary metabolite with a non-growth associated
production. The process to produce penicillin involves an initial batch phase
in which cell growth occurs.
Fermentation Technology—Traditional Processes and Products 265
Beet molasses
Finished mash
storage
Stock inoculum II
Centrifuge
Filtration
IV
Extruders
Culture preparation starts with freeze drying of spores and agar slant
cultures. Vegetative cells are cultivated in the shake flasks and then
transferred to the seed fermenters.
These are agitated tanks of 200500 m3 in volume made of stainless
steel. Mechanical agitation is provided at a rate of 100300 rpm.
Temperature is controlled around 2528°C by using cooling coils.
266 Biochemical Engineering: Principles and Concepts
Spent solvent
Extract I
Purification
Wash of
crystals Filter
Evaporator
Procaine HCI
solution
Filter
Potassium penicillin Mixing
in crystal form tank
Centrifuge
Vacuum
Dryer Product procaine penicillin
The fermentation is stopped when the oxygen uptake rate of the culture
exceeds the oxygen transfer rate of the reactor, or when 80% of the fermenter
is full.
The recovery process includes filtration, extraction, adsorption,
crystallization and drying. Filtration is usually done by using a high
capacity, rotary vacuum drum filters for separation of the mycelia. The
mycelia are washed on the filter and disposed. The penicillin rich filtrate is
cooled to a temperature of 24°C to avoid the chemical or enzymatic
degradation of penicillin.
Solvent extraction is accomplished at low pH values, i.e. at 2.53.0,
using amyl acetate or butyl acetate as a solvent. Two extractors are used in
series and the efficiency of extraction is 99%. Carbon adsorption is used to
remove the impurities from penicillin-rich solvent after extraction.
Crystallization is performed from the solvent or the aqueous phase.
Na, K and penicillin concentrations need to be adjusted along with the pH,
temperature, etc.
Excess amounts of Na or K are added to the penicillin-rich solvent before
crystallization in an agitated vessel.
The crystals are separated by a rotary vacuum filter and are washed and
predried using anhydrous butyl alcohol to remove impurities. Large
horizontal belt filters are used for collection and drying of crystals for which
usually warm air or radiant heat is used.
Final spray drying of the procaine antibiotic precipitate is accomplished
in a low temperature freeze dryer. Biomass may be fully recovered and sold
as an animal food supplement.
Crystalline penicillins G or V are sold as an intermediate or converted to
6-aminopenicillanic acid, used in the production of semi-synthetic penicillin.
Penicillin for medical applications and feed has an annual demand of
$4,400 million. More than 80% of penicillin produced is utilized for the
synthesis of 6-APA and other intermediates.
Vitamins (Vitamin B12): Vitamin B12, is cyanocobalamin, the
deficiency of which leads to pernicious anaemia. The requirement of the
human body for this vitamin is met from diet. Vitamin B12 is produced as
a supplement for animal feeds, for use in pharmaceutical industry, as a food
additive and as a vitamin supplement.
Vitamin B12 is a primary product, commercially produced by
Propionibacterium freudenreichii. The vitamin is extracted from the cells by
a heat treatment.
Most commercially produced riboflavin, vitamin B2 is made chemically
and sold as a vitamin supplement or added to vitamin-enriched bread.
Riboflavin can be produced microbiologically by the yeast Ashbya
gossypii that produces up to 7 g of riboflavin per litre of the growth medium.
Steroids: Cortisone is a well-known steroid and in the production of
this steroid, a strain of bread mould Rhizopus arrhizus is used that is able to
hydroxylate progesterone, an early intermediate in the synthesis of cortisone
by means of microbial hydroxylation.
268 Biochemical Engineering: Principles and Concepts
$TQVJ
/GVJCPQN
Precipitator
2TGEKRKVCVQT
Germ-free
)GTOHTGG
water
YCVGT
5QNWVKQP
HCl
*EN
*[FTQN[\GTU
(KNVGTCKF
/GVJCPQN
/GVJCPQN
%T[UVCNNK\CVKQP
Germ-free
)GTO TGEQXGT[
water
HTGG* 1
/KZKPI
+QPK\GT
'XCRQTCVQT
(KNVGT (KNVGT
5RTC[&T[GT
&GZVTCP
Glucoamylase
Dextrose-rich syrup
Filtration, clarification to
produce 9496% dextrose later
deionized to glucose isomerase
Glucose isomerase
Isomerization to 42%
fructose, HFCS
72% solids
(Refined HFCS)
Secondary refining;
chromatographic fractionation Blending
55% HFCS
and molecular adsorption
9095% HFCS
Figure 8.11 Production of HFCS.
and soluble. The total and soluble protein contents should be lower than
0.3% and 0.03% respectively. This is to avoid colour formation which is a
consequence of the Maillard reaction between amino acids and sugars at high
temperatures.
This is done by saccharifying the liquefied starch slurry using the enzyme
glucoamylase that produces more dextrose from the branched chains of
starch. Glucoamylase is added to the 1015 DE liquefied starch after
temperature and pH adjustment and fed to the reactors. The conditions for
this step are 60°C, and pH of 4.3 with a holding time in reactors of 65 to
75 hours, Finally, the product is said to have 9496% dextrose.
Further, it is refined to remove ash, metal ions and proteins. This step
is essential as proteins and metal ions can interfere in the subsequent
Fermentation Technology—Traditional Processes and Products 271
SUMMARY
In this chapter, we learnt the following:
· Fermentation processes are carried out to manufacture a variety of
industrial alcohols, food products, beverages and fine chemicals.
· Hormones, steroids, alkaloids and vitamins are also manufactured by
fermentation processes.
· Fermentation processes can be aerobic or anaerobic depending upon
the requirements of the product to be manufactured.
· Enzymes and microbial cells are used for the production of all the
fermentation products.
· Yeasts are the preferred organisms for the production of industrial-
scale ethanol.
· Antibiotics like penicillin are produced by using moulds.
· Dextran and xanthan gum are also the products obtained from the
fermentation industry.
· HFCS is a low-calorie sweetner used in beverages, desserts and other
food products.
272 Biochemical Engineering: Principles and Concepts
EXERCISES
8.1 Describe the manufacture of penicillin in detail. Further, how is it
recovered to a purer form?
8.2 With the help of neat flow sheet, describe the production of HFCS and
explain its uses.
8.3 Write a note about the manufacture of the following:
(a) Vinegar
(b) Beer
(c) Rum
(d) Cheese
8.4 Describe the production of citric acid with a neat flow sheet and
indicate its uses.
8.5 Differentiate anaerobic and aerobic fermentation processes with the
help of examples.
8.6 Enlist the microorganisms that are used in a bioprocess industry.
8.7 With a neat flow sheet, describe the production of the following:
(a) Bakers yeast
(b) Acetone
(c) Butanol
Chapter 9
Downstream Processing
Downstream processing refers to the recovery and purification of products of
various biochemical processes. It is bound to be essential to any commercial
process and an important activity in biotechnological industries by means of
which a wide variety of products such as pharmaceuticals, drug intermediates,
food chemicals, beverages, organic fine chemicals and solvents, industrial
enzymes, dairy products, etc. are manufactured. On a wide basis, these are
also called bioseparations.
In downstream processing also called product recovery operations, a
spectrum of techniques, viz. isolation, purification, concentration, etc. are
adopted to bring the desired product from a complex mixture comprising
starting materials, products of reaction, by-products, etc. to a final form
prior to which formulation is done. This envisages the product for long-term
storage, transportation and marketing before it reaches the end users, i.e.
customers. In order to achieve the same, the techniques range from the
conventional chemical engineering unit operations to the sophisticated high
resolution techniques.
Prominently saying, biotechnology without downstream processing is
incomplete.
This chapter emphasizes the use of several conventional and advanced
techniques to isolate the product in a purer form.
9.2.1 Filtration
In the case of filtration (refer to Figure 9.1), rotary vacuum filters, micro-
filters, or ultra filters can be employed depending upon the requirements and
can prove to be cost effective. Figure 9.1 shows the mechanism of filtration.
Fermentation broth is passed through a filter medium over which cake is
obtained on the surface. This medium can act as a septum and hold cake on
its surface. In the case of a rotary drum filter (Figure 9.2), the drum is
covered with a pre-coat layer, i.e. diatomaceous earth. Also filter aids are
used that can enhance the rate of filtration.
Cake formed
Filter medium
Washing jets
Slurry Suction
where dV/dt is the volume flow rate of filtrate and is time dependent.
mÿ is the viscosity of the filtrate, kg/m-sec.
A is the surface area of filter, m2.
DP is the pressure drop through the cake and filter medium and Rm and
Rc with their usual meanings and significance.
Filter medium resistance Rm and its value is usually a characteristic of
filter medium, whereas the cake resistance Rc is seen to increase as filtration
proceeds and later after a start up period Rc supercedes Rm, i.e. the value
of Rc will be greater than the value of Rm.
The value of Rc is expressed as
Rc = a(W/A) (9.2)
where aÿ is the average specific cake resistance, m/kg
W is the total weight of cake formed on the filter, kgs
A is the surface area, m2.
Equation (9.2) can be further expressed as
Rc = a (CV/A) (9.3)
where W = CV and C is the concentration of the cake, i.e. the weight of the
cake formed per unit volume of filtrate, kgs/m3, and V is the total volume
of filtrate.
Substituting Eq. (9.3) in (9.1), we get
D6 '0 ¹ ! ¹ GC
(9.4)
DT ;2M B # ¹6! =N
D 6! '0 ¹ GC
(9.5)
DT ;2M B # ¹6! =N
Integrating the above equation using the boundary conditions as at t = 0,
V = 0 and at t = t, V = V,
6 T
'0 ¹ GC
Ô
D6!
;2M B # ¹6! =N
DT Ô
2M ! È ! Ø
where V0 = and K = É B ¹ # N Ù '0 ¹ GC
B ¹# Ê Ú
i.e.
62M È ! Ø
V2 + A = É '0 ¹ G C T (9.7)
B ¹# Ê B ¹ # N ÙÚ
Equations (9.6) and (9.7) are called Ruth equations for constant pressure
filtration and can be further rearranged as
T
6
+
6 6 (9.8)
Using the above Eq. (9.8) and plotting t/V vs. V by way of which a
straight line is obtained with a slope 1/K and an intercept 2V0/K as shown
in Figure 9.3(a).
V 8
5NQRGQHVJGNKPG-
+PVGTEGRV8-
8
Figure 9.3(a) Plot of t/V vs. V.
a fraction of its surface area will be immersed in the slurry reservoir. This
fraction is represented as f.
The time period during which the filtration is carried out is f/n per
revolution of the drum. Therefore, Eq. (9.6) can be written as
È6 Ø 6 F
ÉÊ ÙÚ 6 +¹ (9.9)
N N N
where V = V¢/n and V¢ is the filtrate volume per unit time and V¢/n is the
volume of filtrate for one revolution of the drum.
During filtration, certain complexities in situation are faced. To simplify
the same, certain assumptions are employed for the analysis of filtration. The
first and foremost is that the cake formed is assumed to be incompressible
although cakes formed after filtration are compressible.
Holding the assumption valid, results in constant specific cake resistance
to be constant, while in reality it is not so and varies with the pressure drops.
It is also found that the use of filter aids, say around 1% to 5%, to
enhance the rate of filtration, also exhibits a significant effect on specific
cake resistance. This effect is illustrated in Figure 9.3(b).
(KNVTCVGXQNWOG
EO HKNVGTCKFKPHGGF
6JKUKUURGEKHKEVQHKNVTCVKQPQH5VTGRVQO[EGU
ITKUGWUDTQVJYJGPVJGR*KUDGVYGGPCPF
6KOG
Figure 9.3(b) Effect of filter aids concentration in the feed on specific cake resistance.
(KNVTCVG
XQNWOGEO R*KU
R*KU
R*KU
6KOGKPOKPWVGU
Figure 9.3(c) pH effects on filtration rate and specific cake resistance (specific to
Streptomyces griseus).
Example 9.1 For the filtration of a cell broth, the constant pressure
filtration was employed. The characteristics of filter employed are:
(i) Area A = 0.3 m2
(ii) Weight of the cake deposited per unit volume of filtrate C = 1950
kg/m3
(iii) Viscosity of the medium m = 3.0 ´ 103 kg/m-s and
(iv) Average specific cake resistance a = 3.0 m/kg.
During the process the following observations were made and recorded.
Estimate the following: (a) Pressure drop across the filter and
(b) Filter medium resistance.
Solution
With the data provided first find out the values for t/V and prepare the
table for t/V and V.
Then plot the graph of t/V vs. V as shown below in Figure 9.3(d).
V 8
5NQRG-s . OKP
+PVGTEGRV8-
OKPNKVTG
8
Figure 9.3(d) t/V vs. V values.
Solution (Hint)
Data: Constant pressure filtration, Area of filter (A) = 1m2; Density of
suspension (r) = 500 kg/m3; Viscosity of filtrate (m) = 1.1 ´ 103 kg/msec.
and the pressure drop DP = 2 bar.
To determine: Filter medium resistance Rm and specific cake resistance a.
282 Biochemical Engineering: Principles and Concepts
Using the tabulated readings calculate t/V and plot the graph of
V vs. t/V as shown in Figure 9.3(d).
Slope of the line =1/K from which get the value of K and the Intercept
= 2V0/K. Using this find the value for V0.
2M ! !
V0 = and K =
B# B# N DP.gc
In the above equations substitute the values and get the Rm and a.
9.2.2 Centrifugation
Centrifugation is taken up whenever the size of the particles is in the range
of 1000.1 µm. It produces a cell-concentrated stream (cream), since the
solids content in a slurry is upto 15% weight by volume. This process is
employed for yeast fermentation recovery. It is used to separate the materials
of different densities when the gravitational force is insufficient for
separation.
In industry, centrifugal force is used (i) to separate fine solids from
liquids by centrifugal sedimentation, (ii) to separate immiscible liquids whose
density difference is small by centrifugal decantation, and (iii) in the
filtration of solids from liquids by centrifugal filtration.
The common types of centrifuges that find application in bioseparations
include the following:
· Tubular bowl centrifuge (Figure 9.4)
· Multi-chamber bowl centrifuge (Figure 9.5)
· Disc bowl centrifuge with/without nozzle (Figure 9.6)
· Decanter centrifuge
· Basket centrifuge (Figure 9.7)
Tubular bowl centrifuge: The tubular bowl centrifuge (Figure 9.4) is the
simplest type and provides high centrifugal force. It has the provision for
cooling and so can be used for protein separation and other products that are
thermally sensitive.
It consists of a long narrow cylindrical bowl suspended from the top
rotating at a high speed, i.e. 10,000 rpm, in an outer casing that is
stationary. The bowl measures from 8 to 15 cm in diameter and upto 150 cm
in height.
Multi-chamber bowl centrifuge: A multi-chamber bowl centrifuge
(Figure 9.5) consists of a number of concentric tubes connected in such a way
that a zigzag flow of the suspension of feed through the chamber is obtained.
Solids get deposited on the outermost chamber wall and solid discharge is
done manually.
Disc bowl centrifuge: The disc bowl centrifuge (Figure 9.6) has a shallow
wide cylindrical bottom driven bowl rotating at a speed of about 6000 rpm
in a stationary casing. The bowl is 30 to 100 cm in diameter and contains
Downstream Processing 283
Liquid
out
r2
Feed
Cake
Length l
Feed r0
r1
Liquid out
Feed
closely spaced metal discs that are located one above the other. The feed is
introduced at the bottom through a centrally located feed pipe from above
and the clear liquid flows out through an annular slit near the neck of the
bowl.
284 Biochemical Engineering: Principles and Concepts
Feed
Denser liquid Lighter liquid
Discs
Solids
r1
r0
r0 = distance of the outer edge from the feed axis
r1 = distance of the inner edge from the feed axis
q = angle at which the discs are fitted.
Figure 9.6 Disc bowl centrifuge.
r0
r2
Slurry Filtrate
r1 Filter medium
Liquid Cake
Q G
FG = Dp3 SP ¹ (9.11)
GC
#$
FD = S F ¹ 5! (9.12)
GC
and
Q G
FB = $
S ¹ (9.13)
P
F
GC
! È Q Ø $P
Ê Ú
It is a well established fact in fluid mechanics that, for spherical particles
when the particle Reynolds number, NRe,p < 0.3, the drag force is then given
by Stokes equation as
&$ QN$P5 (9.14)
GC
Equating Eqs. (9.12) and (9.14), we get
#$
S F ¹5 ! QN$P5 (9.15)
GC GC
After simplifying, we have
N
#$
SF5 $P
or
#$ (9.16)
. 2EP
S F 5 $P
where particle Reynolds number NRe, p =
N
Further, when NRe,p is between 1.0 and 10,000, CD for a sphere is
approximated by
#$ (9.17)
. 2EP . 2EP
or
G:$P SP S
5 #4 (9.20)
N
and
U0,CT = Z U0 (9.21)
RX
i.e. Z = called centrifugal factor while r is the radial distance from the
G
central axis of rotation and w is the angular speed or angular velocity of
rotation and is given as w = 2pNr.
All the above expressions are for dilute suspensions where particle
interactions are ignorable.
For hindered settling that is found in the concentrated suspensions, and
where the particle concentrations are high, the phenomenon is somewhat
complex. The particles interact to form a swarm and consequently their
average velocity decreases from U0 to U.
It is interesting to note that the separation of cells or particles in a
centrifugal field is similar to hindered settling under gravity, since particle
concentration is high under conditions of centrifugation.
In hindered settling, the drag force on the particles is given as
È $P Ø
&$ QN$P5 ÉÊ C , ÙÚ (9.22)
GC
where U is the terminal settling velocity of particles under hindered settling
conditions, L is the average distance between the adjacent particles and b0
is the hindered settling coefficient and is given a value of 1.6 for a
rectangular arrangement of particles.
In dilute solutions, since Dp/L is far lesser than 1.0; we have F¢D » FD.
Therefore, hindered settling becomes more prominent when Dp and L are
comparable.
· Lipid solubilization
· Enzymatic method.
Alkali method: The alkali method is considered to be a cheap and effective
method. Alkali acts on the cell wall including saponification of lipids. Alkali
treatment is carried out at pH of 1112 for about 2030 minutes due to
which proteases, protein splitting enzymes, are inactivated. This method is
found to be more useful in the preparation of pyrogen free therapeutic
enzymes. Pyrogens are the fragments of cell wall of Gram negative bacteria
and when ingest in the body cause increase in the temperature of the body.
By this method enzyme L-asparaginase is successfully isolated.
Detergent solubilization: The solubilization of detergents involves the
addition of a concentrated solution of detergent to about half the solutions
volume of cells to disrupt the cell wall. The process depends on pH and
temperature.
Detergents like sodium dodecyl sulphate (SDS), sodium sulphonate and
sodium taurocholate which are anionic and detergents such as cetyltrimethyl
ammonium bromide (CTAB) which are cationic, and detergents that are
non-ionic, Triton X-100, are employed.
Lipid solubilization or cell wall permeabilization: The solubilization of lipids
or the permeabilization of the cell wall is achieved by the addition of
organic solvents that bring about the cell wall disruption. The solvent is
absorbed by the cell wall resulting in swelling and ultimate rupture. Toluene
is one such organic solvent. The amount of solvent added is 10% of cell
volume. Figure 9.8 depicts the cell wall permeabilization process.
Enzyme digestion: In the case of enzyme digestion, lytic enzymes are added
to the cell suspension. Lytic enzymes being more effective, selective in nature,
gentle and of course very expensive, are employed in some special cases.
With the addition of these enzymes, the digestion of cell wall is
achieved. For example, hydrolyses of a-1, 4-glycosidic bonds in the
mucopeptide moiety of bacterial cell wall is achieved by the enzyme
lysozyme. Yeast cells require a mixture of different enzymes such as
glucanase, protease, mannanase or chitinase.
bead mills and high-pressure homogenizers. These methods find the use in the
laboratory as well as in the industrial scale operations.
A waring blender is particularly effective with animal cells and tissues as
well as with mycelial organisms.
7UGQHHGTOGPVCVKQPDTQVJ
UWDLGEVGFVQHKNVTCVKQPQTEGPVTKHWICVKQP
4GNGCUGQHGZVTCEGNNWNCTRTQFWEVU
9JQNGEGNNUKPUWURGPUKQP
DWVQPQUOQVKEUWRRQTV
OGFKWO
CEVKQPQHN[VKEGP\[OGUCPFEGPVTKHWIKPI
4GNGCUGQHEGNNYCNNRTQVGKPU
2TQVQRNCUV
IGPVNGCIKVCVKQPYKVJEGPVTKHWICVKQP
4GNGCUGQHE[VQRNCUOKEGP\[OGU
1TICPGNNGCPFRTQVGKPRCTVKENG
IGPVNGCIKVCVKQPYKVJEGPVTKHWICVKQP
4GNGCUGQHE[VQRNCUOKEGP\[OGU
2TQVGKPRCTVKENG
GP\[OGU
Figure 9.9 Sequential disruption of cells.
Downstream Processing 291
5QNXGPV5;
*: *:
5KPINGUVCIG
5;
3
8 8 8+ (9.29)
(
8 3+ 8
8 ( 8
8 %8
8 8
Downstream Processing 293
8
8 % (9.30)
;5 ; ; ;0 ;0 5
0
*: : : :0 :0*
P
P
'
P
:P:
Figure 9.10(c) Graph showing the relation between the unextracted solute, extraction
factor and no. of stages in continuous countercurrent extraction.
Also extraction processes can be carried out in two ways: batch and
continuous. The various processes that are envisaged are physical,
dissociative and selective extraction.
For the extraction of biologically active polymers such as proteins,
enzymes and nucleic acids, it is necessary to have a two-phase extraction
system that will not denature the biopolymers. Of course, the two-phase
extraction process is of immense importance in the process of biotechnology.
Aqueous two-phase extraction process: Figure 9.11 depicts the entire
process. The process of extraction involves two steps:
· Mixing the aqueous feed with the two-phase solvent for extraction
· The separation of phases
The very first step in the separation of a desired biopolymer from a
fermentation broth is the removal of cell debris into the heavier phase. The
salt phase in PEG (poly ethylene glycol-potassium phosphate) is a two-phase
system by contacting the aqueous feed with the solvent system.
Biopolymers like proteins enzymes, etc. are extracted into the PEG-rich
lighter phase.
Later, centrifugal separation of two phases is done thereby the lighter
phase is contacted with a solution of the salt at a fixed pH value. The second
stage of extraction isolates other biopolymers, viz nucleic acids,
polysaccharides, etc. leaving purer enzyme or protein in the PEG-rich phase
which is lighter. These two phases are separated by centrifugation.
Next is the purification of the desired protein or enzyme, that is achieved
by a third stage extraction of protein/enzyme into the salt phase by adding
salt solution and changing the pH conditions. It proceeds to give a heavier
salt phase that contains the desired protein at one end and the PEG phase
at the top is a residue that can be recycled. Ultra-filtration is taken up for
the final recovery of the product desired.
Downstream Processing 295
Product recovery
Top phase PEG containing by UF
Figure 9.11 Aqueous two-phase extraction for the recovery of biological polymers.
normal gas and a normal liquid and this is advantageous and is exploited
in the process of extraction. Of course, the changes made in the pressure
values will influence on the properties and alter their behaviour that is from
more gas like to a more liquid like.
In this context, at first we will try to envisage as to what are the critical
parameters, viz. critical temperature and critical pressure?
What is a critical temperature and critical pressure? The critical
temperature of a substance is defined as the temperature above which a
distinct liquid phase cannot exist regardless of the pressure. And the vapour
pressure of the substance at its critical temperature is called the critical
pressure. The other way of defining is, the pressure used to liquefy the gas
at its critical temperature. In general, the substance above and close to its
critical temperatures and pressures will exist as a supercritical fluid. It is
an interesting fact to note that there exist a number of potential extracting
critical solvents that include CO2, N2O, SO2, NH3, C2H6, C3H8, C4H10 and
ethylene. Of these, CO2 is widely used because of its advantages over the
others in terms of being non-toxic and non-hazardous in nature.
The transport properties of a substance at first are to be understood to
decide as to which of these solvents are to be employed. We know that the
transport properties of a solvent as density, viscosity and diffusivity as well
as thermo physical properties as heat capacity and the latent heat of
vaporization are of significance in the extraction processes. Interestingly,
SCFs combine the high solvating power, a characteristic feature of liquids
with low viscosity and high penetrating power or ability, a characteristic
feature of gases.
The physical properties of these SCFs such as viscosity, density and
diffusivity and other similar properties fall intermediate between the
substances in the gases state and in the liquid state. Table 9.1 illustrates the
properties of liquids gases and the SCFs.
From the above table it is clear that the SCFs have low viscosity and
high diffusivity. It is because of this reason the SCFs are used in the
extraction process. It is simply evident that the SCFs due to their low
viscosity and greater diffusivity compared to the liquids diffuse more rapidly
into the substances and even penetrate into the solids. The solvent strength
or the solvating power of SCF can be controlled easily just by increasing the
pressure on the fluid. We know that the increase in the pressure on the fluid
298 Biochemical Engineering: Principles and Concepts
increases the density of the fluid and further the solvent strength. Near the
critical point, small changes in the pressure values will create appreciable
changes in the density of the SCFs.
Also the latent heat of vaporization is found to decrease in the SCF
region and is zero at the critical point and it is due to the fact that there
is absolutely no phase change involved. With all these properties of SCF,
shall enhance the heat capacity as well. It is found that the heat capacity
of SCFs will increase several folds than that of the normal liquid.
Phase diagram: The phase diagram shown in Figure 9.12 is actually a
pressure-temperature diagram also called a P-T diagram of a pure
substance. This is a thermodynamic phase diagram referring to one of the
volumetric properties of a pure substance.
5%(
2%
%2
.KSWKF
2TGUUWTG
5QNKF
62 8CRQWT
6GORGTCVWTG 6%
Figure 9.12 Pressure-temperature diagram of pure substance (CO2).
SCF extraction uses total extraction region (TER) where the density is
between 0.6 and 0.9 and fractionation region (FR) where the density is
between 0.2 and 0.6 grams per cc.
Properties of supercritical fluids: The following are the properties of
supercritical fluids in general.
1. There is a drastic change in some of the important properties of a
pure liquid as its temperature and pressure are increased and
approaches the critical point.
2. The other properties of a liquid change near the critical point as
thermal conductivity, surface tension, viscosity, etc.
300 Biochemical Engineering: Principles and Concepts
6'4
.KSWKF
2 5QNKF
$CTU
(4
2%
)CU
On the other hand, at low solvent power, CO2 exhibits high selectivity
in dissolving the solutes. Thus, supercritical CO2 offers potential scope of
tailoring the extracting parameters in optimizing the recovery and
purification of the desired solute components from a mixture. The solubility
of a solute in super critical CO2 may be enhanced by the addition of a
co-solvent or entrainer. The addition of a co-solvent modifies the thermo-
dynamic affinity between the solute and the extracting solvent favourably.
Co-solvents and Surfactants: Many non-volatile polar substances cannot be
dissolved at moderate temperature in nonpolar fluids such as CO2. For this
reason co-solvent also called entrainers, modifiers, moderators; such as
alcohols and acetone are added to the fluids to increase the solvent strength.
Of late, surfactants are also employed to form the reverse micelles, micro
emulsions, and polymeric latexes in the SCFs including CO2. These
organized molecular assemblies can dissolve hydrophilic solutes and ionic
species such as amino acids, and also proteins. The surfactants that interact
with CO2 are fluoroethers, fluoroacrylates, fluoroalkanes, propylene oxides
and siloxanes.
Schematic diagram of a typical SCFE process unit: Figure 9.14 illustrates
the single stage extraction process that utilizes the feature of adjustability
of the solvent strength with pressure as a variable.
(GGF 'ZRCPUKQP
'ZVTCEVKQP 54
4CHHKPCVG
2TQFWEVQT
%QORTGUUKQP
GZVTCEV
4GE[ENKPIQHUQNXGPV
5QNXGPV
Figure 9.14 Typical SCFE unit.
The feed is given to the extraction chamber to which the solvent is also
fed at relatively high pressure by using a compressor. Within the extraction
chamber the solute/solutes of interest are extracted from the feed. The
products are recovered in the separator by releasing the pressure using an
expansion valve where heating is taking place. The raffinate from the
extraction chamber is collected from the downstream end. From the
expansion valve the solvent is recovered in a solvent recovery system and
the extract also withdrawn simultaneously. The solvent is recompressed and
recycled. The products can also be precipitated from the extract phase by
raising the temperature after the extraction to lower or decrease the solvent
302 Biochemical Engineering: Principles and Concepts
density. Using the profile of increasing pressures, the conditions are set in
such a way that only the lightest components in the feed are extracted in
the first phase. The recovery vessel is then replaced and the pressure
elevated to collect the next heavier fraction.
Using the profile of decreasing pressures with multistage isothermal
conditions, all but the heaviest fraction is extracted in the first vessel. Then
on the extract passes through a series of recovery vessels which are held at
successively lower pressures and each of which precipitates the next lower
molecular weight fraction in the raffinate.
Schematic diagram of SCFE unit using carbon dioxide as a solvent in both
liquid-liquid and liquid-solid extraction: Figure 9.15 can be used both for
the liquid-liquid and solid-liquid extractions. If the feed is L-L, then this
aqueous feed is pumped into the extraction column and the same is made
to contact with the supercritical solvent CO2 at higher pressures (HP). The
solvent is compressed and is recycled into the lower end of the extraction
column. Due to the contact of the feed with the solvent in the first column
the solutes of interest are extracted. The extract phase is then brought to
a lower pressure in a separator still (LP) in order to recover the extract, i.e.
the product from the solvent used.
'ZVTCEVTKEJ%1 'ZVTCEVHTGG%1
UQNXGPV
8CNXG %QORTGUUQT
(GGF
.2 5GRCTCVQTUVKNN
*2 'ZVTCEVQT
4CHHKPCVG 'ZVTCEVQT2TQFWEV
Figure 9.15 SCFE unit using CO2 solvent.
If the feed is S-L, then in that case the solid bed is prepared in the first
column into which the solvent is pumped. This encompasses the leaching of
the solutes from the solid phase and into the solvent phase. Now the extract
is said to be solvent rich, further requires the separation of solvent from it
which is called recovery step. In order to achieve this, extract phase is
pumped to a separator which is held at lower pressures that facilitate the
precipitation of the solid product. Consequently, the solute-lean fluid is then
compressed and recycled.
Downstream Processing 303
%QPEECHHGKPGCPFYCVGT
%CHHGKPGET[UVCNUHTQOET[UVCNNK\GT
Figure 9.16 Schematic diagram of the Kraft process for decaffeination of coffee beans.
In this process the feed comprising moist and mashed coffee beans enter
from the top of the coffee extractor and then carbon dioxide is passed
through the bottom of the extractor. The process of extraction is achieved
within the extractor. The countercurrent flow is preferred over other
configuration as it provides a better contact and the equilibrium attainment
306 Biochemical Engineering: Principles and Concepts
is guaranteed. The decaffeinated green coffee beans are removed from the
bottom of the extractor and are later sent to the roasting tower to produce
coffee while caffeine-rich carbon dioxide is withdrawn as a product from the
first column.
Caffeine-rich carbon dioxide stream now requires the treatment with
water as the separation of CO2 from the caffeine is accomplished. We know
that CO2 is soluble in water to form carbonated water or carbonic acid.
Therefore fresh water is added from the top of the tower through spray
device. Due to this, from the top of the water column the caffeine-lean CO2
is recycled to the first column where it is supplied with make-up CO2. The
downstream end of the second column which is caffeine-rich water is fed to
the reverse osmosis (RO) plant in order to purify the used water so that it
can be reused for the absorption of CO2 and simultaneously concentrate the
amount of caffeine in the product stream by way of membrane separations.
The product stream from the RO plant is concentrated caffeine and to
some extent water. This is later sent to a crystallizer in order to produce
the purer crystals of caffeine.
Overall it is seen that all the 3 separation steps are taking place at high
pressures by using pumps (not shown in the figure).
Note: In commercial practice the dimensions of an extraction vessel are:
diameter is approximately 7 ft. and the height is usually 70 ft.
As indicated earlier, there are several methods proposed for the recovery
of caffeine including washing with water and absorption, but the Kraft
process is widely accepted one. It is apparent that the recovery of a
particular component of an extract is the key challenge in the SCF
extraction process. This is the reason as to why the SCFE is combined with
another process such as distillation, absorption and even adsorption.
7 %1 XCRQWTEQORTGUUQT
'VJCPQNYCVGTHGGF %1GZVTCEVCPV
'ZVTCEVKQPEQNWOP &KUVKNNCVKQPEQNWOPYKVJTGDQKNGT
9CVGTUGRCTCVQT %1VQTGEQXGT[
'ZVTCEVRJCUG 'VJCPQNUGRCTCVQT
%1VQTGEQXGT[ 5VTKRRGT
4CHHKPCVG 'VJCPQN
Figure 9.18 High pressure distillation for separation of EtOH-water mixture using CO2.
While the extract phase is sent to an HPDC from the top to distill CO2
vapours from the overheads and in turn given to the compressor and further
recycled to the extraction column. Ethanol obtained in the bottoms in an
HPDC with traces of CO2 is sent to the recovery unit where even the traces
of CO2 are recovered. Consequently, ethanol is stripped and obtained as a
separate product.
This process is no doubt more complex but said to be versatile in
practice.
By way of SCFE processes it is understood that we can extract up to
95% to 97% by use of a common solvent which is a supercritical fluid carbon
dioxide. With all the advantages understood it is evident that most of the
conventional methods are now replaced by these novel or advanced SCFE
processes.
9.4.3 Sorption
Sorption involves the partitioning of a solute between a bulk solution phase
and a typically porous or high surface area solid. For example, the sorption
of antibiotic streptomycin is done by ion exchange materials.
The ion exchange materials are merely dissociable ion pairs having one
type of charge and being immovable.
For example,
Cation exchanger:
(Solid) H+ + Solute+ (Solid) Solute+ + H+
(Exchanger) (To be removed) (Interaction) (Proton)
Downstream Processing 309
Anion exchanger:
(Solid)+ Cl + Solute (Solid)+ Solute + Cl
(Exchanger) (To be removed) (Interaction)
9.4.4 Precipitation
Precipitation refers to the transformation of soluble solutes into insoluble
solids also called infusible mass which is a precipitate. Here the transformation
means conversion and this conversion is possible because of the decrease in
solubility of the original solute in solvent. This solubility reduction is due to
the addition of a third component. The infusible mass of solid is subsequently
separated from liquid by filtration or centrifugation. It serves as a means of
isolation or recovery and concentration of desired products.
Organic solutes have solubility that depends upon factors like pH of
solution, temperature, composition, ionic strength, etc.
The process of precipitation is brought about in the following ways:
Addition of a precipitant: A precipitant serves to react with a solute and
produces an insoluble product called precipitate, usually a salt. For example,
the recovery of antibiotics is done by adding a precipitant.
Let us see how precipitation is done to isolate penicillin. Penicillin in
a combined form is available as procaine penicillin, sodium penicillin or
potassium penicillin. To isolate pure penicillin, precipitation is done that
separates procaine, sodium or potassium from penicillin.
©0ROCAINE
HYDROCHLORIDE ¸ © 0ROCAINE ¸
ª SODIUMÅBUFFERÅSALT ¹ 0ENICILLIN n ª SODIUM ¹ 0ENICILLIN
ª ¹ ª ¹
« POTASSIUMÅBUFFERÅSALT º «POTASSIUM º
9.5 PURIFICATION
The purification process refers to the removal of contaminating chemicals
from the precipitate formed. This is rather done by taking up the processes
of fractional precipitation, adsorption and chromatography.
This abrupt rise in the solute concentration in the effluent is called break
point at which the concentration is Cb.
The concentration profile in the fluid phase or the variation of solute
concentration in the effluent as a function of time is obtained by plotting
the solution concentration ratio C/C0, versus adsorbent bed length and is
called break through curve.
C is the solute concentration in the liquid at a given time and C0 is the
feed concentration.
The following schematic represents the breakthrough curve for fixed bed
adsorption (Figure 9.19).
%$ %'
++
%%
V$ V5 V'
6KOG
INo adsorption zone; below the curve, and
IIThe mass transfer zone; above the curve
Figure 9.19 Breakthrough curve for fixed adsorption.
After the break point time tB, the concentration abruptly rises to CD or
CE which is the end of the breakthrough curve, where the bed is said to be
saturated.
The break point concentration represents the maximum that can be
discarded in the effluent and is often between 0.010.05 for the ratio CB/C0.
The value of CB/C0 is taken as the point where CD or CE is approximately
equal to C0.
For a narrow mass transfer zone, the breakthrough curve is very steep
and most of the bed capacity is used at the breakpoint. This makes efficient
use of the adsorbent and hence lower costs for regeneration.
Once the breakpoint is reached, the feed is stopped and the adsorbed
material is eluted using different solvents or pH or ionic strength conditions.
When the entire bed comes to saturation with the feed, the total capacity
of the bed may be taken to be proportional to the area under the curve and
#
the line = 1.0.
#
The total capacity or the time equivalent to the total capacity or the
stoichiometric capacity of the bed is given as
È # Ø
TTOTAL Ô ÉÊ # Ù DT
Ú
(9.35)
The usable capacity of the bed is given by the breakpoint time tB. The
time tU is the time equivalent to the usable capacity or the time at which
the effluent concentration reaches its maximum permissible limit and usually
is very close to the tB. It is given as
È # Ø
T5 Ô ÉÊ # ÙÚ DT (9.36)
Now the ratio tU/ttotal, the fraction of the total bed capacity or the
length of bed utilized up to the breakpoint, HB is given as
È T5 Ø
(" É Ù (4 (9.37)
Ê TTOTAL Ú
where HT is the total length of the bed.
The length of unused bed HUNB is then the unused fraction of the total
length and is given as
(5." T5 T (4
TOTAL
(9.38)
concentration in the feed is high, all contribute to the shape of the curve
produced.
The breakpoint is sharply defined in some case and in others poor.
Generally, the breakpoint time tB, decreases with decrease in the bed height,
increase in the particle size of an adsorbent, increased flow rate of the fluid
through the bed and increased initial solute content of the feed.
9.5.3 Chromatography
Chromatography, a separation technique, refers to a group of closely-related
techniques that are quite useful in the analysis of chemical substances. These
techniques find use in the separation, purification and identification of
compounds before quantitative analysis is taken up.
The basis of separation in any of the chromatographic techniques is the
selective distribution of the components of a mixture between two
immiscible phases in intimate contact with each other. One of these phases,
called stationary phase, is a solid or an immobilized liquid (i.e. a liquid
coated on a finely-divided inert solid). The other phase is called mobile
phase (eluent or carrier gas depending on whether it is a liquid or gas) and
percolates through the stationary phase. The sample to be analyzed is
usually dissolved in the mobile phase.
Figure 9.20 shows the classification of chromatographic techniques.
Chromatography
PC TLC
GSC GLC LSC LLC
GLC: Gas-Liquid Chromatography; GSC: Gas-Solid Chromatography; LSC: Liquid-Solid
Chromatography; LLC: Liquid-Liquid Chromatography; PC: Paper Chromatography;
TLC: Thin Layer Chromatography
Figure 9.20 Classification of chromatographic separations.
Column chromatograph: Figure 9.21 represents the Column chromato-
graphic separation of a two-component mixture say A and B.
Sample mixture is introduced along with a compatible solvent phase at
the head of column which is packed with a stationary phase. The components
Downstream Processing 315
Legend: A and
B
Figure 9.21 Chromatographic separation of a two-component sample mixture.
start distributing themselves between the two phases. Once the adsorption is
complete, the mobile phase is added continuously, with a constant flow rate
at the head of the column. With this, there is a redistribution of the solute
components with a simultaneous migration down the column along with the
mobile phase. Series of such distributions occur between the mobile and
stationary phases leading to different rates of migration of solutes A and B.
It leads to separate bands which elute from the column at different times.
Online chromatogram: Figure 9.22 represents an online chromatogram,
which shows different resolutions of peaks detected with respect to different
times.
Retention time (tR): Retention time is defined as the time taken by the
solute to reach the detector from the moment it is injected into the column.
It is determined by measuring the distance between the sample injection
point to the apex of the peak on an on-line chromatogram.
Planar chromatography: Planar chromatography (Figure 9.23) uses a strip
of filter paper instead of a column with an adsorbent in it.
The stationary phase is immobilized water held by cellulose molecules
of the filter paper and the mobile phase is a homogenous liquid. It consists
of a glass jar with a lid within which is placed a Petri dish called eluent
reservoir. A small amount of sample is spotted at one end of the filter strip
using a capillary or a hypodermic syringe. It is placed inside the jar,
thereafter a chromatogram develops.
The solute components of the sample move up the paper based on
their distribution coefficients between the stationary and the mobile
phases. Different solutes of the mixture will move to different extents and get
316 Biochemical Engineering: Principles and Concepts
tR1
X
tR2
tm A
Injection
Y
X-axis = detector signal
Y-axis = time for elution of components
tm = the time for the mobile phase to flow through the
column and retain in the column
tR1 = retention time of the solute eluting in peak 1.
tR2 = retention time of the solute eluting in peak 2.
A and B = components in a sample mixture to be analyzed.
Lid
Solvent front
Filter
paper Sample
spots
dm and ds = the linear distances of mobile and solute phases respectively from the starting point.
%QWPVGTNKICPF
.KICPF
5RCEGTCTO
#EVKXGUKVG
/CVTKZ
Figure 9.24 Ligand-counter ligand interactions.
Downstream Processing 321
The coupling of a ligand also has the influence over the interactions
between. The steps involved in the coupling or pairing of chosen ligand to
the selected matrix are as follows.
1. Chemical activation of the matrix, rather than going for thermal
activation as this is thermally labile.
2. Immobilization of ligand via the chosen functional groups as
mentioned above.
3. Blocking or deactivating the residual active groups that do not take
part in the interactions.
In general, the proper selection of support matrix, ligands and selection
of spacer arms will lead to an efficient binding and interactions that later
provide the values of K, an equilibrium constant in the moderate range as
mentioned in the above paragraphs.
This value of K (104 to 108) shall provide a reversible and at the same
time a proper interaction between the ligand and counter ligand so that
finally the separations and purifications are efficient.
In the succeeding paragraphs, is described the actual mechanism by
showing the conceptual representation of affinity chromatography and the
generation of on-line chromatogram (Figure 9.25).
'NWVKQPQHFGUKTGF
0QPTGVCKPGFEQORQPGPVU EQORQPGPVU
9CUJKPI
5CORNG 'NWVKQP
&'6'%6145+)0#.XU6+/'
The biggest advantage of this process is that the aroma and the flavour
of the foodstuffs is not lost, although it is an expensive process.
During lyophilization, it is seen that the material will be composed of
a frozen core and as and when ice sublimes, i.e. transformation of solid ice
directly into vapour form takesplace, the plane of sublimation recedes from
outer surface thereby leaving a porous structure of the material. The heat
of sublimation which is about 2800 kJ/kg of ice is conducted inward through
the layer of dried material and the water vapour is also transferred through
the layer of it. Thus it can be seen as a simultaneous heat and mass transfer
during such process. This vapour pressure difference required for sublimation
is attainable by maintaining total pressure in the drying chamber and should
be of the order of 0.1 to 2.0 torr. In addition to it, a condensing system is
also provided to remove water vapour formed along with heating system to
provide the required latent heat of sublimation to the frozen material.
Equipments like batch freeze driers are employed in practice and are
realized the following added advantages. These are porous structure of
substance which is due to the removal of water by sublimation, shape and
size retained, shrinkage and case hardening are almost negligible and above
all the heat damage is minimized.
9.6.2 Formulation
Formulation is required particularly for pharmaceuticals in the form of
tablets, capsules, injectibles, creams, powders, etc. Also it is needed in other
products like, bakers yeast, industrial proteins and enzymes, etc. depending
upon their end uses.
Formulation of a product should ensure the product stability, and more
importantly the requirements of the consumer or end user. The succeeding
lines with examples illustrate the importance of formulation step.
As we know bakers yeast is produced by aerobic fermentation process
and is later concentrated by centrifuging followed by filtration using rotary
vacuum filter to a dry matter content of about 35%. Later this concentrated
yeast is extruded into long filamentous mass segmented to pieces and
subjected to drying using fluidized bed mechanism employing temperatures
between 40 to 60°C. This dried yeast formulation is stable and amenable for
long-term storage and transport. Before using it is soaked in water which
helps in restoring its biological activity.
In all the biochemical processes that make use of enzymes called
biocatalysts for continuous operation of industrial processes, these enzymes
are needed to be formulated or immobilized on suitable supports or matrices
for long-term stability and use inside the packed bed or fluidized bed
reactors.
Various techniques are discussed in Chapter 5 that describe the
importance of immobilization of enzymes. The following examples with
applications shall depict the importance of enzyme formulation.
Downstream Processing 327
Retentate recycling
Feed tank
Permeate or Product
Figure 9.26 Membrane separation plant.
Membrane modules
(a) Flat sheet: These are easy to construct and also are amenable to
replacement of membranes whenever required. The channel width formed
between the membranes can be altered to reduce the plugging problems
during the process. In between is placed a support that helps to operate at
higher pressures.
These are also called flat-plate systems. Their features include low
membrane surface-to-volume ratio. Feed is given from one side and the
product permeates exit from the other.
330 Biochemical Engineering: Principles and Concepts
Feed
Membranes
Retentate Permeate
Support
(a) Flat sheet Feed
Axial flow of feed
Permeate Spacer
Membranes
Support and
spacer
Feed Permeate
Shell flow
(b) Spiral wound
Membrane
Tubes
Permeate
Retentate Hollow fibres
(c) Shell Shell
Feed
Retentate
Permeate
(d) Hollow fibre
Figure 9.27 Different membrane modules.
(c) Shell and tube: Shell and tube membrane modules resemble shell
and tube heat exchanger modules. In these, the membranes are placed on the
tube side and the feed is given on the shell side. They are also called tubular
membranes.
The characteristic features are as follows:
The packing density of shell and tube membrane modules is 30200 m2/m3.
Their resistance to fouling is very good and it is easy to maintain. Cost per
square metre of membrane surface is low to moderate. Their pressure drops
is very low and can be used for high pressure applications such as reverse
osmosis, ultra filtration and gas permeation processes.
The capillary flow model: Figure 9.28(a) depicts the capillary flow model
and the features of this model are mentioned below:
(i) Membrane is considered to be loose and microporous and capable
of retaining the solutes or particles of sizes larger than 10 A°.
(ii) The flow of feed occurs through the pores and mainly due to the
convective flow, except in an impermeable layer.
(iii) A filtering or sieving type mechanism occurs wherein the solvent
moves through the micropores in essentially a viscous flow and the
solute molecules small enough to pass through the pores are carried
by convection with the solvent.
332 Biochemical Engineering: Principles and Concepts
(iv) The passage of the larger molecules is prevented by the size of the
micropores.
(v) Capillary flow dominates in micro-filtration and ultra-filtration
processes.
/KETQRQTQWUNC[GT
QHOGODTCPG
2QTGRTQXKFKPICUKGXKPI
OGEJCPKUO
5QNWVGUNCTIGT
VJCP
In Figure 9.28(a) it is seen that the pores are available for the transport
of solutes through the membranes. Overall it is seen that the permeants are
transported by pressure driven convective flow through tiny pores.
The separation takes place due to the permeants getting excluded from
some of the pores in the membrane through which the other permeants move.
It is seen that in the microporous Knudsen flow gas separation
membranes, transport occurs by pore flow or capillary flow.
The solution diffusion model: The features of this model are mentioned
below:
1. This is applicable to the membranes which are non-porous or dense
in structure.
2. There is always the diffusion of solutes or molecular species to be
transported in the membrane material and subsequently follows the
molecular diffusion across the barrier following the Fickian type of
diffusion.
3. It is due to the applied pressure, a concentration gradient is set up
that acts as a driving force.
4. The permeants are separated because of the differences in the
solubilities of the materials in the membrane and also the differences
in the rates of migration or the rates at which the materials diffuse
through the membrane.
Downstream Processing 333
&GPUGOGODTCPG
/GODTCPG
$QWPFCT[NC[GT ,,U
%O
,%
%D %R
Z ZE
Figure 9.28(c) Boundary layer film model.
On rearranging, we have
D# *S
DX (9.44)
# # P $
Integrating the above equation, using the limits at x = 0, C = Cb and at
x = d, C = Cm, we get
È# M # P Ø
Js = k¢ ln É Ù where k¢ = D/d the mass transfer coefficient.
Ê #B # P Ú
(9.45)
Here if no solute passes through the membrane, then we get,
È# Ø
Js = k¢ ln É M Ù (9.46)
Ê #B Ú
The ratio Cm/Cb increases if the flux Js is high or k¢ is low. The low
values of k¢ may be due the following reasons related to one another. They
are the low values of D, diffusivity which is due to the high molecular weight
or due to the high viscosity of the solution or also due to the large values
of d, the boundary film thickness seen due to the low magnitude of
turbulence. Indeed, the values for k¢ are to be determined empirically.
Feed Retentate
(Slurry)
Semi-permeable
membrane
Permeate
Figure 9.29 Flow diagram of reverse osmosis.
P>p
Pure solvent II
Solution Membrane
I III
p is osmotic pressure
Figure 9.30 Osmosis and reverse osmosis.
Figure 9.31(a) illustrates the process taking place across the membrane
which is considered to be thin film composite or asymmetric membrane.
.KSWKFHGGF
2GTOGCVGQH
RTGUUWTG2
RWTGYCVGT
RTGUUWTG2
9CVGT
+PQTICPKEUCNVU
EQNNQKFUQTICPKEU
/1UDNQEMGFD[
OGODTCPGU
.KSWKF
TGVGPVCVG
2 2
The feed is a liquid at a high pressure P1, containing solvent (water) and
solubles, viz. inorganic salts, colloidal matter, etc. No sweep liquid is used,
but the other side of the membrane is maintained at a much lower pressure
P2. The membrane being perm selective, for the solvent, as acetate or
aromatic polyamide can be employed.
In order to withstand the large pressure drops, the membrane must be
thick for which composite structures or asymmetrical membranes that have
a dense skin or layer on a thick porous support is used.
340 Biochemical Engineering: Principles and Concepts
/GODTCPG
,9YCVGTHNWZ
CPF
,5UCNVHNWZ
, 9
(GGF ,5 2TQFWEV
EQPEGPVTCVG RGTOGCVG
UQNWVKQP UQNWVKQP
% %
2 2
$S +S
*S # # (9.49)
,M
or can be written as
Js = As (C1 C2) (9.50)
The terms in the above equations refer to the following:
Ds is diffusivity of solute in membrane m2/sec
Ks is Cm/C, the ratio of concentration of solute in the membrane to the
concentration of solute in the solution. It is also as good as partition
coefficient or distribution coefficient and Lm is the membrane thickness in
metres.
From Eq. (9.50) it is seen that C2 is usually lower than C1 as the salt
concentration in permeate is less. Therefore, we can write the above
equation as
Js = As (C1) (9.51)
and this is independent of P.
From the equations it is evident that the water flux is proportional to
the applied pressure, but the salt flux is independent of pressure.
%YR 5MKP
2QTQWU %YH
UWRRQTV
%UO
%UR
causes mass transfer of salt by diffusion from the membrane surface back to
the bulk feed.
The back rate of salt diffusion depends upon the mass transfer coefficient
for the film or boundary layer on the feed side. The lower the value of mass
transfer coefficient, the higher is the value of Csi.
This Csi has a greater significance as it fixes the osmotic pressure and
thus influences the driving force for water transport through the membrane.
In general, it is seen that due to the onset of concentration polarization
in the RO, membranes develop fouling and overall the performance is
affected.
The major applications of RO in wastewater management are as follows:
· Desalination of industrial wastewater after the secondary treatment
· Water recovery in a dye house effluent
· Recovery of water from domestic sewage for industrial use
· In pollution control, for the removal of certain hazardous organic
pollutants present in wastewater.
9.7.2 Ultra-filtration
The process of ultra-filtration is operated in the range of 210 bar pressure.
The separation process occurs across the membrane that discriminates solute
molecules on the basis of their size. Figure 9.32 shows a flow diagram of
ultra-filtration.
Feed Retentate
(Slurry)
Microporous
membrane
Permeate
(GGFVCPM
/GODTCPGOQFWNG
(GGFRWOR
%QPEGPVTCVG
%QNNQKFCNQTRCTVKEWNCVGU
$WNMUQNWVKQP
5WTHCEG
HQWNKPI
+PVGTPCN
HQWNKPI
%
IGN
,
X
, %
X K
,
Y
%
KD
, , %
U X K
& F%
K K
FZ
At any point within the boundary layer, the convective flux of solute to
the membrane surface is given by volume flux, Jv of the solution through the
membrane multiplied by the concentration of the solute Ci.
At steady state, this convective flux within the laminar boundary layer
is balanced by the diffusive flux of the retained solute in opposite direction
as shown in Figure 9.33(c).
D#
* V# I $ (9.55)
DX
where Di is the diffusivity of macromolecules viz. proteins in the boundary
layer. Once a gel layer is formed the concentrations of solute at both the
surfaces of the boundary layer are fixed. At one surface the concentration is
the feed solution concentration Cib and at the other it is the concentration
at which the solute forms an insoluble layer of gel, i.e. Cgel.
Integrating Eq. (9.55) using the boundary conditions as at x = 0,
C = Cib and at x = d, C = Cgel, we get
E # GEL
È *V Ø D# I
É $ Ù DX
Ê IÚ Ô Ô #I (9.56)
# IB
# GEL È* ¹ E Ø
EXP É V Ù (9.57)
# IB Ê $I Ú
9.7.3 Micro-filtration
Micro-filtration is also called microporous filtration or cross-flow filtration
(Figure 9.34). Though it resembles conventional filtration, the filter medium
being a microporous membrane, it is capable of filtering particulate matter
or suspended solids rather than dissolved solute molecules.
Retentate
Feed
(Slurry)
Microporous
membrane
Permeate
Figure 9.34 Flow diagram of micro-filtration.
9.7.4 Dialysis
Dialysis is also called hemodialysis in the medical field, as it is required to
purify the blood of the patients suffering from renal failure. It also has
potential applications in the field of bioseparations, for example, separation
of alcohol from beer.
Figure 9.35 shows a flow diagram of dialysis. Dialysis involves the
separation of solutes by diffusion across the membrane from one liquid phase
to another liquid phase, on the basis of molecular size and the molecular
weight (100 < MW < 500).
Purified
Feed stream
Impurities Dialysate
solution
Membrane
Legend:
Anode
_
+
Cathode
_
+
9.7.5 Nano-filtration
Nano-filtration is also a type of membrane separation process based on size,
solution diffusion, etc. The pores of membranes of nano-filtration are smaller
in size as compared to those of ultra-filtration membranes. Therefore, most
of the organic compounds barring very low molecular linear chain organics
are rejected while monovalent cations combined with monovalent anions
from a compound or salts pass through the permeator.
The membranes employed in nano-filtration are low pressure RO
membranes with very high rejections and high permeates of salt at low
concentrations, but lose their selectivity at salt concentrations above
10002000 ppm of salt in water. For this reason, membranes are used to
remove low levels of salt from already relatively clean water. They operate
at very low pressure of 50200 psig.
Major applications of nano-filtration are as follows:
1. Treatment of dairy wastewater
2. Decolouring of spent mineral acids
3. Removal of heavy metals from industrial effluents
4. In paint industry, dye industry, pulp industry, etc.
9.7.6 Electrophoresis
Electrophoresis is a method of separation of charged molecules applying an
electric field. This technique of separation was first enunciated by A.
Tiselius in the year 1937.
It refers to a technique which involves the movement of charged
particles or ions in an electric field resulting in their migration towards the
oppositely charged electrodes, i.e. when the charged molecules are placed in
an electric field they migrate towards the oppositely charged electrodes.
This migration depends upon the net charges, size, shape and the
applied electric potential.
It is more a technique that is normally used in the separation of charged
biomolecules.
Downstream Processing 351
$QWPFCT[$
$WHHGTUQNWVKQP
.QYGTKPI 4KUKPI
2TQVGKPUQNWVKQP $QWPFCT[:
JCXKPIEQORQPGPVU
:;<
$'(14'
%QPEITCFKGPV
< $WHHGTUQNWVKQP
.QYGTKPI
: 4KUKPI
:
;
#(6'4
;
< :
;
<
Figure 9.37 Location of protein components in an electrophoresis cell before and after
application of electric field.
$WHHGT
5CORNG
(TCOG
#)#415')'.
The resolution is much higher in this technique. The other gels in use
are polyacrylamide, sodium dodecyl sulphate (SDS) etc.
2TQVGKPOKZVWTG .CTIGUK\GFRQN[RGRVKFGU
5OCNNUK\GF
RQN[RGRVKFGU
/KITCVKQPQH
RTQVGKPU
The proteins are denatured and have a negative charge with a uniform
charge to mass ratio when treated with SDS reagent. This SDS reagent is
a surfactant and reduces the surface tension of the components in question.
Proteins migrate towards anode at alkaline pH through PAGE gel
during the process. The smaller peptides move faster followed by the larger
polypeptides.
Therefore, the inherent charge on proteins is dampened in SDS-PAGE.
Consequently, the separation is based on the size.
Downstream Processing 355
Intracellular
Pure enzymes: In solution phase or can be enzymes:
generated as finished product. Finished
product.
SUMMARY
In this chapter, we learnt the following:
· The recovery and purification of products at the end of the
fermentation process is an essential step.
· The product separation can be accomplished either integrated with or
next to the fermentation step.
· Separation of biomass or other insoluble products, concentration or
primary isolation of product and purification are the major categories
of separation.
· For the product biomass, the separation strategy is relatively simple
and involves filtration and centrifugation followed by coagulation/
flocculation of the cells.
· For soluble products like antibiotics, enzymes, organic acids, etc., the
separation strategy depends on whether the products are extracellular
or intracellular.
· The separation strategy is simpler for extracellular products as
compared to the intracellular products as cell disruption is not
required.
· Proteins require a special strategy for separation such as isoelectric
focusing or electrophoresis.
· Membrane, adsorptive, or extraction separation schemes can be used
for the simultaneous separation of products during fermentation
process.
· Affinity chromatography is one in which one or more components in
the sample mixture to be analyzed show an affinity or attraction
towards their counterparts. This makes the process overall highly
specific and efficient compared to the others.
· Gradient elution involves the change of eluent composition during the
development either continuously or stepwise, so that the partition
coefficient values of each component are changed with respect to time.
· Crystallization operates at low temperatures, and certainly minimizes
the threat of thermal denaturation and degradation of the so, called
heat labile materials.
· The two models to understand the transport mechanisms in
membranes are:
Capillary flow model or pore flow model and solution diffusion model.
· Electrophoresis refers to a technique which involves the movement of
charged particles or ions in an electric field resulting in their
migration towards the oppositely charged electrodes, i.e. when the
charged molecules are placed in an electric field they migrate towards
the oppositely charged electrodes.
360 Biochemical Engineering: Principles and Concepts
EXERCISES
9.1 Discuss the importance of downstream processing in a bioprocess
industry.
9.2 Explain in detail the physical methods of cell separation.
9.3 What is cell permeabilization? What is its significance?
9.4 Discuss the steps involved in the product isolation and purification of
an enzyme.
9.5 What are the advantages of liquid-liquid extraction? Give an account
of the theoretical principles and steps involved in the aqueous two-
phase extraction of an enzyme.
9.6 What is supercritical fluid extraction? Discuss the principles and the
characteristics of the same.
9.7 Enlist the advantages of supercritical fluid extraction.
9.8 Give a schematic diagram of a membrane separation unit and identify
the components.
9.9 With principle explain in detail the following membrane separation
processes:
(a) Electrodialysis
(b) Dialysis
(c) Ultra-filtration
(d) Reverse osmosis
(e) Micro-filtration.
9.10 Give an account of the steps involved in large scale precipitation of
proteins.
9.11 Give a broader classification of chromatographic separation techniques.
9.12 Describe the chemical and enzymatic methods of cell disruption.
9.13 Write a note on the following:
(a) Thermolysis
(b) Osmotic shock
(c) Ultrasonication
Downstream Processing 361
general, one may view the possible sites of action of an antimicrobial agent
by recalling certain features of the microbial cell.
The manner in which the antimicrobial agents inhibit or totally destroy
the microorganisms can be attributed to the following actions:
1. Cell wall is damaged or cell wall synthesis is inhibited.
2. The permeability of cytoplasmic membrane is altered.
3. Proteins and nucleic acids and their physical states are altered.
4. Enzyme action is inhibited.
5. Synthesis of protein and nucleic acid is totally prevented.
0 100.0
5 109.0
10 115.0
15 121.5
20 126.5
Steam inlet
in chamber
Temperature sensor
and jacket
Drain
Figure 10.1 Autoclave.
steam chamber equipped with devices that permit the chamber to be filled
with the saturated steam and maintained at the designated temperatures and
pressure for any period of time. In the operation of an autoclave, it is
absolutely essential to remove the air and replace it by steam. This is
because, the presence of air will reduce the temperature obtained within the
chamber substantially below that which would be realized if pure saturated
steam were under the same pressure. In reality, it is not the pressure of the
steam, but the temperature of the steam that kills the microorganisms.
Various media, discarded cultures, solutions and contaminated materials
are the materials that are routinely sterilized by using an autoclave.
366 Biochemical Engineering: Principles and Concepts
For instance, the agar slant cultures of some bacteria, yeasts, and moulds
are normally stored for longer periods at a refrigeration temperature of about
47°C. Many bacteria and viruses are maintained at deep freeze temperatures
that range from 20 to 70°C.
Microorganisms maintained at the freezing or the sub-freezing
temperatures may be considered dormant; they perform no detectable
metabolic activity. Of course, this condition is useful in the preservation of
food materials. From the practical point of view, high temperatures may be
considered as microbicidal and low temperatures as the microbistatic.
10.4.3 Desiccation
Desiccation refers to the cessation of the metabolic activity of a microbial
cell. It is followed by the decline in the total viable population of the
microorganisms. The process depends upon the following factors:
1. The microorganism type, i.e. whether filamentous or non-filamentous,
bacterial origin, etc.
2. The material in which the organisms are dried.
3. The totality of drying process.
4. The physical conditions to which the dried organisms are exposed. The
conditions are light, temperature, and humidity.
10.4.5 Radiations
In the context of radiations, the most significant radiations are the
electromagnetic radiations, for example, light rays and X-rays. Gamma rays
are more penetrating than any other rays.
When such radiations are passed through cells, they create free hydrogen
radicals, hydroxyl radicals and some peroxides, which in turn can cause
different types of intracellular damage. Due to this, the ionizing radiations
are not that specific. This is called the method of cold sterilization. Hence,
it can be used in the sterilization of temperature sensitive materials like food
materials and pharmaceuticals.
Even sound waves are used in the process of sterilizing biological
materials.
10.4.6 Filtration
Seitz filters having asbestos, Berkefeld filters having diatomaceous earth,
ChamberlandPasteur filters, etc. are some of the biological filters that are
used in the removal of microorganisms from gases and liquids.
In these devices it is not a mere ordinary filtration that can be seen as
mechanical sieves and porosity is not the only factor preventing the passage
of organisms, but there are also other factors like the electric charge of the
filter, the electric charge carried by the organisms, and the nature of the fluid
being filtered that can influence the efficiency of the filtration process.
Membrane filters are used more recently and extensively both in the
laboratory and in the industry to sterilize materials. It is customary to force
the fluid through the filter by applying a negative pressure to the filter flask
by use of a vacuum or the water pump or to impose a positive pressure above
the fluid in the filter chamber, thus forcing it through.
On the completion of the filtration, precautions are taken to prevent the
contamination of the filtered material when it is transferred to other containers.
Of late, more advanced filters are developed called High Efficiency
Particulate Air (HEPA) filters that produce dust and bacteria free air.
Applications of physical agents for the control of microorganisms are
summarized in Table 10.2.
Using moist heat: For sterilizing the following: Cannot be used for heat-
Autoclave is the device. Utensils, surgicals, culture labile or sensitive materials
media and other liquids. and is found to be ineffective
against the organisms inside
the materials impervious to
steam.
Use of fibre glass filter Used in the process of air Very expensive process.
(HEPA) disinfection.
No single agent is the best for any and all purposes. As a result, several
classes of chemical compounds and substances have been identified that have
destructive effects in terms of their suitability for practical applications.
the growing forms but not the resistant spore forms of disease-producing
microbes. Disinfectants are normally used on inanimate objects.
Antiseptic: An antiseptic is a substance that opposes the sepsis. It prevents
the growth or action of microbes either by destroying them or by inhibiting
their growth and metabolism.
Sanitizer: A sanitizer refers to an agent that reduces microbial population to
safe levels as judged by the public health requirements. It usually kills
growing bacteria to the extent of about 99.9 per cent. Sanitizers are usually
applied to inanimate objects. They are also applied in daily care of
equipment and utensils in dairies, food plants, restaurants, etc. Sanitization
implies with the sanitary condition while disinfection does not necessarily
imply.
Germicide (Microbicide): A germicide is an agent that kills the growing
forms but not necessarily the resistant spore forms of germs. In practical
sense, a germicide is the same as a disinfectant. For any application,
germicides are commonly used for killing all kinds of germs.
Bactericide: A bactericide is an agent that kills bacteria.
Fungicide: A fungicide is an agent that kills fungi.
Virucide: A virucide is an agent that kills viruses.
Sporicide: A sporicide is an agent that kills spores.
Bacteriostasis: Bacteriostasis refers to a condition in which the growth of
bacteria is prevented.
The agents that have in common the ability to inhibit the growth of
microbes are collectively called microbistatic agents.
Antimicrobial agent: An antimicrobial agent is the one that interferes with
the growth and metabolism of microbes. There are certain antimicrobial
agents that are used to treat infection and are called chemotherapeutic agents.
Of the above, heating and chemical addition methods are widely used in
large-scale processes. Sensitive vitamins and complex molecules are sterilized
by passage through porous membranes.
The requirements for destruction of viable microbes and viruses vary
widely upon the material and its intended uses. For example, in the biological
treatment of wastewater, using trickling filters or activated sludge process;
microbes are naturally present in the process fluid and are responsible for
desirable reactions. But in the manufacture of alcohol, vinegar and silage,
inhibitors for the growth of unwanted microorganisms are rapidly evolved.
Here also the sterilization requirements are not extreme. In the
pasteurization of milk, all the microorganisms are not killed. In fact, severe
treatment leads to degradation.
On the contrary, in pure culture fermentation, tissue culture, food
products, and canning industry, stringent sterilization requirements are to be
met with. Essentially all contaminants are to be excluded. But the degree of
perfection may vary. The degree of perfection is based on the Contamination
Probability (CP), an index used to measure the sterilization. If CP (1P0)
is of 102; (where P0 = extinction probability during sterilization), then it
can be taken for batch fermentation. On a large scale, one batch out of
hundred is lost due to contamination, it means, sterilization requirements
though are stringent, they cannot be met totally. So (1P0) is the fraction
of sterilizations expected to fail to produce a contaminant-free product.
Sterilization can be taken up in batch or continuous fashion. For batch
processes, the methods are as follows:
· Steam sparging
· Electrical heating
· Steam (Heat exchanger)
· Coolant (Heat exchanger)
The only merit that we find here is these are relatively simple processes.
The demerits are as follows:
· Time needed for heating and cooling is more.
· Extent of thermal damage to desirable compounds.
· Vitamins are destroyed by heating.
· Proteins denature at elevated temperatures.
Therefore the sterilization process should operate at the Highest Feasible
Temperature and for the Shortest Time (HTST) that is necessary for the
microbial death.
Medium to be
sterilized To vacuum
Expansion valve
Throttling device
Flash cooler
Holding section
Sterile medium
(a) Continuous injection type
Medium sterilized
Steam
Holding section
SUMMARY
In this chapter, we learnt the following:
· Microorganisms can be removed, inhibited or killed by various
physical agents, physical processes or chemical agents.
· The major physical agents or the processes used for the control of
microorganisms are high temperature, low temperature, desiccation,
osmotic pressure, radiations and filtration.
· Sterilization refers to the complete elimination of microorganisms
while disinfection does not.
· The major groups of antimicrobial agents are phenols and phenolic
compounds, dyes, halogens, detergents, heavy metals and their
compounds aldehydes, etc.
· Large-scale bioreactors place heavy demand on processes to sterilize
fluids entering the bioreactor.
· Liquid streams are thermally sterilized or filter sterilized. Steam
sterilization is preferred, but the process must not damage the ability
of the medium to support the growth.
· Continuous sterilization protects the medium components from
degradation better than batch sterilization.
· Air streams are typically filter sterilized. Gases are sterilized by using
surface filters which are of membrane cartridge type.
· There are two types of continuous sterilization reactorscontinuous
injection type and continuous plate exchanger type.
EXERCISES
10.1 Define the following terms:
(a) Sterilization
(b) Disinfection
(c) Lyophilization
(d) Desiccation
(e) Sanitizer
The Control of Microorganisms 377
10.2 What are the conditions that influence the antimicrobial action?
10.3 Explain the mode of action of antimicrobial agents.
10.4 Describe the control of microorganisms by using chemical agents.
10.5 Write about the control of microorganisms by physical agents.
10.6 Explain the process of fractional sterilization.
10.7 Write about the characteristics of antimicrobial chemical agents.
10.8 What are the major groups of chemical antimicrobial agents?
10.9 Write a short note on the following:
(a) Dry heat
(b) Incineration
(c) Osmotic pressure
(d) Disinfectant
10.10 Discuss sterilization reactors in detail.
Appendix A
Process Kinetics and
Reactor Analysis
379
380 Appendix A Process Kinetics and Reactor Analysis
2 First order
1
1
1
Zero order
Log (Concentration)
Figure A.1 Reaction order.
C0
slope = k
C (conc. of
reactant
remaining at
time t)
Time, t
Figure A.2 Zero order reaction.
C0
Conc. of
reactant at
time t Slope of tangent = dC/dt
Time, t
Figure A.3 First order reaction.
A P
D#
K# K# (A.5)
DT
Unit of k, which is reaction rate, is per time (time–1).
Integrating Eq. (A.5), we get
È# Ø
LN É Ù KT
Ê# Ú
or
È# Ø
LN É Ù KT
Ê# Ú
or
È# Ø KT
LOG É Ù
Ê# Ú
Appendix A Process Kinetics and Reactor Analysis 383
Plotting log C versus time t, we get the graph as shown in Figure A.4.
log C
slope = 0.434 k
Time, t
2A P
D;!=
R K;!=
DT
If C is the concentration of A at any time t, then
D#
R K# (A.6)
DT
where k = reaction rate constant and is given as volume/mass-time.
Rearranging and integrating Eq. (A.6), we get
D#
KDT
#
KT (A.7)
# #
384 Appendix A Process Kinetics and Reactor Analysis
Plotting the above equation, i.e. 1/C versus time t, we get the graph as
shown in Figure A.5.
1/C
slope = k
1/C0
Time = t
È D# Ø È D# Ø
6 É !Ù 6 É !Ù 6 K#!
Ê DT Ú NET Ê DT Ú REACTION
or
È D#! Ø È D#! Ø
ÉÊ DT ÙÚ ÉÊ DT ÙÚ
NET REACTION
or
D#
K#
DT
Integrating the above equation, we get the final form after rearrangement
as
È# Ø
T LN É Ù
K Ê #D Ú
C0
V Ce
Ce
D# Ø D# Ø
6 ÈÉ 1# 1#E 6 ÈÉ
Ê DT ÙÚ NET Ê DT ÙÚ REACTION
Appendix A Process Kinetics and Reactor Analysis 387
or
D# Ø
6 ÈÉ 1# 1#E 6K#E
Ê DT ÙÚ NET (A.8)
D# Ø
At steady state, ÈÉ
Ê DT ÙÚ NET
#E
# È6 Ø
É ÙK
Ê1 Ú
È# Ø
T#&342 É Ù (A.11)
K Ê #E Ú
Q
C0
V C1
C1
V C2
C2
#
First reactor: (A.12)
# KT#&342
#
Second reactor: (A.13)
# KT#&342
N
# È Ø
É Ù (A.14)
# Ê KT#&342 Ú
N Ë È # Ø N Û
NT#&342 ÌÉ Ù Ü (A.15)
K ÌÍ Ê # N Ú ÜÝ
Appendix B
Bioenergetics
Glycolysis, the process of breakdown of glucose for the production of pyruvic
acid using the path ways, and the Krebs cycle are some of the processes which
are part of bioenergetics.
Bioenergetics deals with energy-related processes in organisms. In order
to obtain kinetic energy, all organisms carry out catabolic processes in which
organic substances are broken down into either inorganic substances or
simpler organic compounds having lower level of energy status. The
phenomenon of liberation of energy from organic substances in a catabolic
process is called respiration. The process of respiration also provides
important intermediates that are required in the synthesis of several
important organic compounds.
The energy released during respiration is available for the synthesis of
ATP and ADP and inorganic phosphate, and the process is called oxidative
phosphorylation.
Respiration can be divided into two formsaerobic respiration and
anaerobic respiration, In aerobic respiration the usual mode or mechanism is
considered while in anaerobic respiration, also termed fermentation it is
obligatory in only a few microorganisms. During this process the respiratory
substrate usually glucose is incompletely oxidized to form the intermediates,
later producing ethanol and carbon dioxide.
B.1 METABOLISM
We know that the life support activity of even simple organisms involves a
set of complex biochemical reactions. One such complex biochemical reaction
is the process of metabolism.
Metabolism is a process that is the sum total of such biochemical reactions
taking place inside the living cell. It is a combination of two different
processes, one called catabolism and the other anabolism (Figure B.1).
389
390 Appendix B Bioenergetics
ATP
Oxidized
ADP ANABOLISM precursors
biomass
NADP NADPH
Oxidized products,
CATABOLISM CO2
NADH
ENERGY GENERATION
H2 O Oxidative phosphorylation O2
ATP Substrate level phosphorylation ADP + P
Figure B.1 Metabolism processes with energy generation.
B.2 CATABOLISM
Glycolic pathway for the production of pyruvic acid from glucose can be
taken as the best example of catabolism. Most organisms oxidize
carbohydrates of which the most common is glucose, which is usually an
energy source. Respiration and fermentation are the two processes taken up
by organisms in producing energy from glucose molecules. Both respiration
and fermentation start with the glycolic pathway wherein glucose is broken
down to pyruvic acid in series of steps as shown in Figure B.2. Glycolysis is
also called EMP pathway.
Glycolysis or EMP pathway is common to both kinds of respiration and
takes place in the cytoplasm of the cell. It consists of two steps.
Step 1. Conversion of carbohydrate or glucose to fructose-1,6-
diphosphate also biphosphate
Appendix B Bioenergetics 391
Glucose
2ATP
2ADP
Fructose biphosphate
2-Triose phosphate
2NADH
2NAD+
2ADP
2-Pyruvic acid
2-Phosphoglyceric acid
2ATP
Glucose
Fructose biphosphate
2 Lactic acid
2-Triose phosphate
2ATP 2ADP
2NADH2 2NAD
2-Phosphoglyceric acid
2-Pyruvic acid
2ADP 2ATP
Figure B.3 Transformation of pyruvic acid to lactic acid.
Glucose
2 Ethanol
Fructose biphosphate 2NADH2 2NAD
2 Acetaldehyde
2-Triose phosphate
2ATP 2ADP
2NADH2 2NAD
2-Phosphoglyceric acid
2-Pyruvic acid
2ADP 2ATP
Figure B.4 Pyruvic acid to ethanol (alcoholic fermentation).
Glucose Fumarate
Pyruvate Succinate
Oxaloace
Citrate tate Succinyl CoA
Cis-aconitrate a-oxaloglutarate
Isocitrate Oxalosuccinate
Glucose 1,3-Diphosphoglycerate
1 6
3 5 8
Dihydroxyacetone 4 Pyruvate
phosphate
Legend:
1. Hexokinase
2. Glucose phosphate isomerase
3. Phosphofructokinase
Glyceraldehyde 4. Triose isomerase
3-phosphate 5. Glyceraldehyde 3-phosphate
dehydrogenase
6. 3-Phosphoglycerate kinase
7. Phosphoglyceratemutase
8. Enolase
9. Pyruvate kinase
Figure B.6 Pentose phosphate pathway.
394 Appendix B Bioenergetics
B.3 ANABOLISM
Anabolism is the process where the released energy is utilized to synthesize
new molecules. The molecules that can be synthesized are proteins from
amino acids, carbohydrates, i.e. from sugars to polysaccharides, etc.
The metabolic oxidation of glucose to carbon dioxide and water is
considered effective, while some amount of energy produced is lost as heat
and rest is utilized for anabolic processes.
Exon Intron
Gene
Transcription
mRNA precursor
Nucleus
Cytoplasm
Polypeptide chain
Region to be
studied DNA probe
Double-stranded
DNA
Denature
Single-stranded DNA
Anneal Primer
Immunize mice
Harvest antibody-producing
lymphocytes
Screen immunoglobulin-producing
hybridomas
Harvest
Purification
Disease
HLA Industrial Microbiology Virology susceptibility
purification testing
Figure B.11 Applications of MABs and DNA probes.
Appendix C
Concepts in Environmental
Microbiology
C.1.1 Rickettsias
These are said to be ordinary Gram-negative bacteria and are obligate
parasites. The parasitic features show that they truly depend on the others
that are normally referred to as the hosts. These grow within the host cells
that are said to be disease causing or pathogenic.
400
Appendix C Concepts in Environmental Microbiology 401
C.1.2 Mycoplasmas
Mycoplasmas are ordinary Gram-negative bacteria and are said to belong
the order Mycoplasmatales and the family Mycoplasmataceae.
The distinctive features of mycoplasmas are as follows:
1. They lack cell walls that can certainly act as shape giving structures
to the microbes. But they do have outer cytoplasmic covering or
membrane that predominantly helps in the ingress and the egress of
the nutrients.
2. Shapes are not rigid or rather flexible as they lack cell walls. They
can assume different shapes and forms from spheres to the branched
filaments.
3. These are susceptible to lysis by osmotic shock, that is, sudden
change in the cell nutrient and the surrounding conditions may be due
to dilution of the cell suspension or medium with water.
4. Even higher levels of penicillin cannot inhibit as they lack cell walls.
However, they are inhibited by antibiotics like tetracyclines and
chloramphenicols that can influence in one way or the other the
process of protein synthesis.
5. They have the ability of cultivating in vitro on the non-living media
as facultative anaerobes or obligate anaerobes.
402 Appendix C Concepts in Environmental Microbiology
6. They also have parasitic features and mucous membranes, and the
joints of animals are the regions that host these microbes. For their
growth, they require cholesterol and can cause various diseases like
pneumonia and urogenital disorders.
C.1.3 Archaeobacteria
Archaeobacteria are the bacteria with unusual properties, one of the four
categories as stated earlier.
These can be Gram-positive or can be Gram-negative. The important
features are as follows:
1. Archaeobacteria are phylogenetically different from eubacteria.
2. Methanogenic or methane-producing bacteria, extreme halophiles and
thermoacidophiles are the three types of archaeobacteria which have
a tendency to withstand extreme acidic and high temperature
conditions.
3. Methane producers require high levels of NaCl for growth while others
grow at a low pH and at a high temperature.
Generally, they are employed in the treatment of wastes that generate
methane gas as a fuel.
As referred to section 1.7, environmental microbiology pertains to the
study of microbes that interact with the various means of environment as
soil, water and air. Their reactions, influence, physical and chemical changes
that can be brought upon due to some kind of transformations are the real
concepts of environmental microbiology. Also it has been stated that it refers
to the study of microbiology of water, soil, and aquatic systems, domestic
and industrial wastewater. In this discussion, is also included their
distribution in nature, relationship to each other and to the other living
organisms. We know that microorganisms are closely associated with health
and welfare of humans; but some being beneficial and some detrimental.
Environmental microbiology provides an insight to the study of
microorganisms in a broader perspective and a broader spectrum that
encompasses overall features like their metabolic activity, growth, aging
processes and altogether their life processes when applied in the field of
environmental engineering; may be air, water and soil pollution. It has been
realized that one can modify the conditions of the environment to assess the
metabolic activities, growth patterns and even their details of genetic
pattern. All these can be done without destruction of a microbe. An
environmental engineer should also assess that well before the
microorganisms are exploited for various commercial applications in the
treatment of wastes, etc. It is quite essential to acknowledge the basics of
microorganisms and their features so that use of microbes is a boon and not
a curse. All these concepts are dealt with in Chapter 1.
Characterization, classification and identification of microorganisms are
Appendix C Concepts in Environmental Microbiology 403
Basically, antigens are the chemical compounds of microbial sources, and this
antigenic feature has a great influence and a practical significance. An
antigen reacts with an antibody.
Stability is the criterion to asses the situation that is more proned to the
frequent and radical changes, perhaps leading to a sort of confusion. In this
regard, every attempt is made to devise classifications that need only minor
changes as and when new information is available.
· Intuitive method
· Numerical taxonomy, and
· Genetic relatedness.
5VTCKP:5VTCKP;
5VTKPIUQH&0#
FQWDNGUVTCPFGF5VTKPIUQH&0#
FQWDNGUVTCPFGF
6JGTOCNFGPCVWTCVKQP6JGTOCNFGPCVWTCVKQP
5GRCTCVKQPQH&0#UVTCPFU5GRCTCVKQPQH&0#UVTCPFU
#PPGCNKPIVQIGVJGTVQCRRTQRTKCVGVGORGTCVWTG
1WVEQOG (QTOCVKQP QH JGVGTQFWRNGZ VYQ UVTCKPU CTG ENQUGN[ TGNCVGF KH
JGVGTQFWRNGZKUPQVHQTOGFVYQUVTCKPUCTGPQVENQUGN[TGNCVGF
*GVGTQFWRNGZ+VTGHGTUVQCUVTCPFQHQPGQTICPKUOVJCVRCKTUYKVJCUVTCPF
QHVJGQVJGTQTICPKUO
Figure C.1 DNA homology technique.
The selective method is the most preferred one over others as it is much
more specific to a particular microorganism. In the selective method of
isolation, we find that there is favour made to the desired species in terms
of growth while discouraging or even killing the other microorganisms
present in the mixed culture.
Also there are certain other methods available like chemical methods of
selection, physical methods of selection and even biological methods of
selection. The use of dilute media, with very low levels of carbon or nitrogen
sources as growth medium, favouring the growth of a specific microorganism
and use of inhibitory chemicals like bile salts, dyes, salts of heavy metals,
etc. are the chemical methods, while heat treatment methods, use of
incubation temperature and use of pH medium are the physical methods.
Exploiting the advantage of pathogenic properties of microorganisms and
using animal as the selective medium in which the microorganism can infest
and grow is the biological method of selection.
In all probability, by using one or the other method, the isolation of pure
cultures can be done. There are also several known methods or techniques to
isolate pure cultures. They are as follows:
· Streak-plate technique
· Pour-plate technique
· Spread-plate technique
· Roll-tube technique
· Micromanipulator technique
observe the features of colonies and the broth cultures. The colony
characteristics are determined by the features such as size, texture, elevation,
consistency, opacity and translucency called optical properties, pigmentation
and colour of the colonies. The characteristics of the broth cultures are also
required. The amount of growth which may be scanty, moderate or abundant
and the distribution and type of growth, whether uniform, uneven, confined
to the surface of the broth as a scum or pellicle or even sediment that can
get accumulated during the process are all the characteristics of cultures to
be determined.
Mixing tank
Raw water reservoir Pump Alum addition
Sedimentation
Coagulation
descends through the water in the settling basins. Then the water is passed
through the sand filters in which 99% of bacteria are removed.
Subsequent to this water is sent for disinfection by the addition of
chlorine or by means of ozonation or also by using UV radiations. This is
to done to ensure the potability of water. Chlorine dosage must be sufficient
to leave a residual content of 0.2 to 2.0 mg per litre free chlorine.
Besides this, the purification processes may also include the techniques
for removing the minerals that cause hardness to water, pH adjustment if
water is too acidic or alkaline. With all these procedures performed, water
is now said to be potable.
C.10 BIODEGRADATION
Biodegradation refers to the use of microorganisms or their metabolites to
treat toxic chemicals and render them harmless. There is a growing faith that
once developed and proven, the process of the biodegradation is potentially
less expensive than any other approach to neutralize toxic wastes. This is
said to involve a low capital investment, low energy consumption and often
self-sustaining operations.
In biodegradation, a microbe feeds on the toxic water, or in the presence
of a particular waste, it secretes an enzyme to degrade the waste and also
reduces the volume of organic matter. Today, microorganisms that degrade
some of the most toxic chemicals are being isolated from the soil of
hazardous waste sites. Later these organisms can be selectively breed in the
laboratory and returned to the environment as more efficient scavengers.
Biodegradation techniques are versatile and can be used at different
stages. Some of the potential applications of biodegradation include
removal of contaminants from raw materials before processing, treatment of
pipe line wastes before discharge from the installation of a factory,
decontamination of soils and surface and ground water and the clean-up of
dump sites. Biodegradation operations can be carried out in a number of
ways.
1. Microorganisms or their active metabolites can be released into the
environment which is contaminated.
2. Already being present in the environment, can be enhanced by the
addition of suitable nutrients that favour the growth and their
activities called bioaugmentation.
3. Microorganisms to be used in contained or semi-contained reactors.
Biodegradations are applied for herbicides and pesticides to reduce their
concentration levels. Oil biodegradation is also an important application of
this process. In this case oil spills and oil seeps, and their effects are reduced
gradually by sequential metabolism of various classes of compounds present
in oil. It is said that a species of bacteria Pseudomonas putida feeds on the
contents of various hydrocarbon products like xylene, camphor, etc. and
reduce the effects of oil spillage especially in water.
Appendix C Concepts in Environmental Microbiology 413
Self-Assessment Exercises
124. In the multiple lag phases during growth multiple carbon sources are
utilized by the micro organisms. (True/False)
125. Yield coefficients for organisms growing aerobically on glucose range
between
(a) 0.9 and 1.4 g/g
(b) 0.4 and 0.9 g/g
(c) 0.9 and 1.0 g/g
(d) none of these
126. CSTRs or CSTFs can be operated as turbidostats which refer to the feed
metered in such a way that
(a) biomass concentration decreases
(b) biomass concentration is constant
(c) biomass concentration increases
(d) none of these
127. At high dilution rates it is seen that the substrate concentration and
biomass concentration at steady state
(a) increases and decreases respectively
(b) decreases and increases respectively
(c) both increase
(d) both decrease
128. Specific rate of production is also called cell productivity. (True/False)
129. As dilution rate D nears maximum specific growth rate mmax and X
becoming infinitesimally small, this condition is called ..............
.............................
130. High fructose corn syrup (HFCS) is a low calorie sweetener with a
higher level of fructose (55%). (True/False)
131. Prednisone, cortisone, dexamethasone, corticosteroids commonly known
as steroids are manufactured by using the raw material, complex
alcohols. (True/False)
132. Dextran is commercially produced using an organism Leuconostoc
mesenterodes. (True/False)
133. Primary metabolites as amino acids, nucleotides and proteins are
produced during the growth phase and are needed for growth. (True/
False)
134. Antibiotics are generally secondary metabolites. (True/False)
135. A decanter centrifuge is also called ..............................................
136. In the process of cell disruption, this is not a physical method.
(a) thermolysis
(b) osmotic shock
(c) lipid solubilisation
(d) ultrasonication
Appendix D Self-Assessment Exercises 427
Glossary
Acclimation: Automatic adjustment of microorganisms to the surroundings.
Acetyl choline: Most common neurotransmitters.
Acetyl Co A: The entry compound from the Krebs cycle in cellular
respiration, formed from a fragment of pyruvate attached to a coenzyme.
Actin: A globular protein that links into chains, two of which twist helically
about each other to form micro-filaments in contractile muscles.
Active site: Specific part of an enzyme that attaches to the substrate by
means of weak chemical bonds.
Aerobic respiration: Harvesting chemical energy in the form of ATP from
food molecules, with oxygen as an electron acceptor.
Allosteric site: A specific receptor site on an enzyme molecule away from the
active site.
Alpha (a) helix: Form of secondary structure of proteins arising from a
specific hydrogen bonding giving a spiral shape.
Amino acid: An organic molecule having both carboxyl and amino groups,
and are monomers of proteins.
Anaerobic respiration: Respiration that occurs in a few groups of bacteria
leaving in anaerobic environments such as soil. In this case sulphates and
nitrates are the electron acceptors.
Antibiotic: A chemical that kills or inhibits the growth of bacteria, usually
by transcriptional or translational regulation.
Antibody: An antigen binding immunoglobulin produced by B cells and
functions as effector in an immune response.
429
430 Glossary
Cellular respiration: The most efficient catabolic pathway for the production
of ATP.
Cellulose: Structural polysaccharide of cell walls consisting of glucose
monomers held by glycosidic bonds.
Chitin: Structural polysaccharide found in many fungi and in the
exoskeletons of all arthropods.
Cholesterol: A steroid forming an essential component of animal cell
membranes and can act as a precursor molecule for the synthesis of other
important steroids.
Coenzyme: An organic molecule serving as a cofactor. Many vitamins
function as coenzymes in important metabolic reactions.
Cofactor: Non-protein molecule that is needed for proper functioning of an
enzyme.
Competitive inhibitor: A substance that reduces the activity of an enzyme by
competing with a substrate.
Cyanobacteria: Photosynthetic, oxygen producing bacteria also called blue
green algae.
Cyclic AMP (cAMP): Cyclic adenosine monophosphate.
Cytoskeleton: Network of micro-tubules, micro-filaments that serve a variety
of mechanical and transport functions.
Denaturation: A process in which a protein loses its native conformation,
thereby becoming biologically inactive.
DNA: Deoxyribonucleic acid, double stranded and can replicate in
transmission of characters.
DNA polymerase: An enzyme that catalyzes the elongation of new DNA
during replication.
DNA probe: A chemically synthesized, radioactively labelled segment of
nucleic acid used to find a gene of interest.
Enzymes: A class of proteins serving as catalysts.
Essential nutrient: A chemical element required by animals and plants and
cannot be synthesized within itself.
Exons: The coding regions of a eukaryotic gene that are expressed and are
separated from each other by introns.
Fat (Triacylglycerol): A biological compound consisting of three fatty acids
linked to one glycerol molecule.
Fatty acid: A long carbon chain carboxylic acid.
432 Glossary
Bibliography
435
436 Bibliography
Index
Acetone-butanol production, 262 Animal cells, 17
Actinomycetes, 15 Antibiotics, 264265
Activation energy, 66 Apoenzyme, 64
Active site, 87 Arrhenius equation, 66
enzymes, 63 Ascospores, 14
Adenine, 43 Asparaginase, 38, 126
Adenosine diphosphate (ADP), 390 Aspartate, 126
Adenosine triphosphate (ATP), 430
Adsorption, 310
Aerobic fermentation (see processes) B-1, 4 glycosidic bond, 55
Aerobic organisms, 10 Bacillus, 11
Aerobic processes, 11, 263 Bacteria, 1011
bakers yeast production, 264265 Bacteriophages, viruses, 3, 16
citric acid production, 263 Bakers yeast production, 264265
high fructose corn syrup, 268 Balanced growth, 173
penicillin production, 264267 Batch and continuous reactors, 243244
Agarose, 57, 138 Batch cultivation, 219
Airlift reactor, 248 Batch growth, 179, 185
Alanine, 38 batch culture, 179
Algae, 16 death phase, 179
Allosteric effectors, 100 decline phase, 181
Allosteric enzymes, 113 defined, 179
Allosteric inhibition, 100 exponential growth phase, 179
Allostery, 100 growth patterns and kinetics in, 179
Amino acids, 36 lag phase, 179
chemical structure of, 36 logarithmic phase, 179
Amylase, 126 logistic equations, 192
Amylopectin, 56 phases of, 179
Amylose, 56 stationary phase, 179
Anabolism, 394 Batch kinetics, 179
Anaerobic fermentation, 255 Bioaffinity, 318
acetone-butanol production, 262263 Biological catalyst, 63
ethanol production, 259261 Biomass holdup, 166
lactic acid production, 257258 Biomass production, 169
437
438 Index